Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: MTMR4, a phosphoinositide-specific 3'-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

doi: 10.1111/gtc.12609

Figure Lengend Snippet: FIGURE 6 MTMR4 knockdown inhibits starvation-induced increase in dephosphorylated TFEB/total TFEB ratio. (a) Phosphorylation of TFEB and other proteins and total amounts of MTMR4 and MTMR3 in control and MTMR4-depleted cells. A549 cells transfected with control- or MTMR4-siRNAs were placed in growth medium (fed) with or without 100 μM chloroquine or in HBSS for 4 hr, and subjected to western blot analyses. S6K, S6 kinase. (b) Quantified data of total TFEB protein level (sum of the dephosphorylated and phosphorylated forms of TFEB) (left), phospho-TFEB level (normalized to GAPDH) (middle) and ratio of dephosphorylated TFEB/total TFEB (right) in control and MTMR4–depleted cells. (c) Time course of starvation-induced change in phosphorylation of TFEB and S6K. A549 cells were starved by culture in HBSS for the indicated time periods and subjected to western blot analyses. Stv, starved. (d) Phosphorylation of TFEB in control and MTMR4-depleted cells under both fed and starved conditions with or without cyclosporine A treatment. Control and MTMR4-depleted A549 cells were incubated in growth medium (fed) or HBSS (starved) with or without cyclosporine-A (10 μM) for 4 hr

Article Snippet: GFP- TFEB expression vector (# 38119) was provided by Dr. Shawn Ferguson through Addgene (Roczniak- Ferguson et al., 2012).

Techniques: Knockdown, Phospho-proteomics, Control, Transfection, Western Blot, Incubation

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: MTMR4, a phosphoinositide-specific 3'-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

doi: 10.1111/gtc.12609

Figure Lengend Snippet: FIGURE 8 PI(3)P-phosphatase MTMR4 regulates endocytic and autophagic pathways. MTMR4 is localized mainly in LEs and APs to regulate PI(3)P levels. MTMR4 regulates the fusion, fission, motility and maturation of endosomes, the autophagic pathway, and lysosomal biogenesis. MTMR4 is also required for activation of TFEB. This action of MTMR4 contributes to MTMR4 regulation of the endocytic and autophagic pathways

Article Snippet: GFP- TFEB expression vector (# 38119) was provided by Dr. Shawn Ferguson through Addgene (Roczniak- Ferguson et al., 2012).

Techniques: Activation Assay

Journal: Cell Death Discovery

Article Title: Inhibiting BCKDK in triple negative breast cancer suppresses protein translation, impairs mitochondrial function, and potentiates doxorubicin cytotoxicity

doi: 10.1038/s41420-021-00602-0

Figure Lengend Snippet: A Quantification of BCKDK , BCKDHA , PPM1K , BCKDHB , BCAT2 , and BCAT1 mRNA expression corrected to 18S/HSPCB reference genes in MCF10A, BT549, and MDA-MB231 cells. B , C BCAA catabolic enzyme expression in TNBCs treated with 2 µM DOX for 18 h. Quantification of BCKDK , BCKDHA , PPM1K , BCKDHB , BCAT2 , and KLF15 mRNA expression corrected to 18S/HSPCB reference genes in MDA-MB231 ( B ) and BT549 ( C ) cells. D Immunoblot and densitometric analysis of BCKDK, total and phosphorylated BCKDHA E1α Ser 293, DLD and KLF15 in MDA-MB231 cells treated with 1 and 2 µM DOX for 18 h. Quantifications are from three independent experiments. Statistical analysis was performed using a two-way ANOVA followed by a Tukey’s multiple comparison test; * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated. *MCF10A vs. BT549 or DMSO vs. 1 µM DOX, #MCF10A vs. MDA-MB231 or DMSO vs. 2 µM DOX. E , F UPLC MS/MS analysis of intracellular BCKAs in E MDA-MB231 and F BT549 cells. All data are presented as mean ± S.D. Statistical analysis was performed using a Student’s t test; * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated.

Article Snippet: Adenoviral vector expressing BCKDK shRNA (shADV-225358) and the control vector for scrambled shRNA GFP (Cat# 1122) were obtained from Vector Biolabs (USA).

Techniques: Expressing, Western Blot, Comparison, Tandem Mass Spectroscopy

Journal: Cell Death Discovery

Article Title: Inhibiting BCKDK in triple negative breast cancer suppresses protein translation, impairs mitochondrial function, and potentiates doxorubicin cytotoxicity

doi: 10.1038/s41420-021-00602-0

Figure Lengend Snippet: A Immunoblot and densitometric analysis of BCKDK, total and cleaved Caspase 3, cleaved Caspase 7, total and cleaved PARP, total and phosphorylated ATM Ser 1981 in MDA-MB231 cells transfected with siCON, siBDK#1, or siBDK#2 for 72 h followed by 2 µM DOX or DMSO for 18 h. B , C MDA-MB231 ( B ) and BT549 ( C ) cells were transfected with siCON, siBDK#1, or siBDK#2 for 72 h followed by 2 µM DOX or DMSO treatment for 18 h and analyzed for Caspase 3 activity. D , E MDA-MB231 cells transfected with siCON, siBDK#1, or siBDK#2 for 72 h followed by 2 µM DOX or DMSO treatment for 18 h and analyzed for D LDH release in the media, and E metabolic activity measured by CCK8 assay. Quantifications are from three independent experiments. A – D , *within groups (DMSO vs DOX), #between DOX groups (siCON vs siBDK#1 or vs siBDK#2), E , *siCON vs siBDK#2, #siCON+DOX vs siBDK#1+DOX, @siCON+DOX vs siBDK#2+DOX. Data presented as mean ± S.D. Statistical analysis was performed using a two-way ANOVA followed by a Tukey’s multiple comparison test; * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated.

Article Snippet: Adenoviral vector expressing BCKDK shRNA (shADV-225358) and the control vector for scrambled shRNA GFP (Cat# 1122) were obtained from Vector Biolabs (USA).

Techniques: Western Blot, Transfection, Activity Assay, CCK-8 Assay, Comparison

Journal: Cell Death Discovery

Article Title: Inhibiting BCKDK in triple negative breast cancer suppresses protein translation, impairs mitochondrial function, and potentiates doxorubicin cytotoxicity

doi: 10.1038/s41420-021-00602-0

Figure Lengend Snippet: A Immunoblot and densitometric analysis of total and phosphorylated BCKDE1α Ser 293, BCKDK, total and cleaved Caspase 3, total and cleaved PARP, total and phosphorylated ATM Ser 1981 in BT549 cells pre-treated with 125, 250, and 500 µM BT2 for 20 h followed by 2 µM DOX or DMSO treatment for 18 h. B Measurement of Caspase 3 activity in BT549 cells pre-treated with 100 µM and 150 µM BT2 for 20 h followed by 2 µM DOX or DMSO treatment for 18 h. Caspase 3 activity was measured in MDA-MB231 ( C ) and BT549 ( D ) cells and LDH release into the media was measured in MDA-MB231 cells ( E ) pre-treated with 500 µM BT2 for 20 h followed by 2 µM DOX or DMSO treatment for 18 h. Quantifications are from three independent experiments. A , *within groups (DMSO vs DOX), #between DOX groups (siCON vs siBDK#1 or vs siBDK#2); C – E , *within groups (DMSO vs DOX), #between groups (VEH vs BT2 and VEH+DOX vs BT2+DOX). Data presented as mean ± S.D. Statistical analysis was performed using a two-way ANOVA followed by a Tukey’s multiple comparison test; * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated.

Article Snippet: Adenoviral vector expressing BCKDK shRNA (shADV-225358) and the control vector for scrambled shRNA GFP (Cat# 1122) were obtained from Vector Biolabs (USA).

Techniques: Western Blot, Activity Assay, Comparison

Journal: Cell Death Discovery

Article Title: Inhibiting BCKDK in triple negative breast cancer suppresses protein translation, impairs mitochondrial function, and potentiates doxorubicin cytotoxicity

doi: 10.1038/s41420-021-00602-0

Figure Lengend Snippet: A Principle component analysis plot for all gene expressions from MDA-MB231 cells treated with both siRNAs targeting BCKDK. B Distance matrix clustering for all gene expressions from MDA-MB231 cells. C Heatmap for differentially expressed genes from MDA-MB231 cells. D Top 20 most significantly enriched gene ontology (GO) terms from BCKDK knockdown induced differentially expressed genes. Color represents the fold enrichment statistic, and size represents the percentage of the differentially expressed genes in the gene set compared with the total gene set size. E Heatmap for genes involved in apoptosis which were significantly differentially regulated by BCKDK knockdown in MDA-MB231 cells.

Article Snippet: Adenoviral vector expressing BCKDK shRNA (shADV-225358) and the control vector for scrambled shRNA GFP (Cat# 1122) were obtained from Vector Biolabs (USA).

Techniques: Knockdown

Journal: Cell Death Discovery

Article Title: Inhibiting BCKDK in triple negative breast cancer suppresses protein translation, impairs mitochondrial function, and potentiates doxorubicin cytotoxicity

doi: 10.1038/s41420-021-00602-0

Figure Lengend Snippet: A Heatmap for genes involved in mitochondrial biogenesis, structure, and function significantly differentially regulated by BCKDK knockdown. B Immunoblot and densitometric quantification of complex I–V proteins in MDA-MB231 whole cell lysates transfected with siCON, siBDK#1, or siBDK#2 for 72 h. Data presented as mean ± S.D. Statistical analysis was performed using a two-way ANOVA followed by a Tukey’s multiple comparison test; * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated. C – H Mitochondrial function measured in BCKDK depleted MDA-MB231 cells using extracellular flux analyzer in the presence of 25 mM glucose. C Basal OCR, D ATP production measured in MDA-MB231 cells transfected with siCON or siBDK#2. E Basal and maximal OCR, F ATP production, G spare capacity, and H proton leak measured in MDA-MB231 cells treated with 250 or 500 µM BT2 for 20 h. B , *siCON vs siBDK#1, #siCON vs siBDK#2; C – D , *within groups (DMSO vs DOX), #between groups (siCON vs siBDK#2); E – H , *VEH vs 250μM BT2, #VEH vs 500μM BT2. Data presented as mean ± S.D. Statistical analysis was performed using a was performed using Student’s t test; * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated.

Article Snippet: Adenoviral vector expressing BCKDK shRNA (shADV-225358) and the control vector for scrambled shRNA GFP (Cat# 1122) were obtained from Vector Biolabs (USA).

Techniques: Knockdown, Western Blot, Transfection, Comparison

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis.

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: Figure 2. Conditional Deletion of TFEB in OL Lineage Cells Causes Ectopic Myelination in the Cerebellar Molecular Layer (A and B) Genetic labeling reveals OPCs in the P14 mouse cerebellar molecular layer (red in A and B, indicated by white arrowheads). ML, molecular layer; GL, granule layer.

Article Snippet: GFP-TFEB expression construct was generated by subcloning mouse TFEB cDNA into a pEGFP-N1-Flag vector, which was a gift from Patrick Calsou (Addgene plasmid, #60360) (Britton et al., 2014).

Techniques: Labeling

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis.

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: Figure 4. TFEB cKO Mice Harbor Thicker Myelin Sheaths and Are Precociously Myelinated in White Matter (A and A’) Representative TEM micrographs from P21 Ctl (TFEBF/F) and TFEB cKO (Olig2-Cre; TFEBF/F) CC showing that myelinated axon density is increased in the absence of TFEB. (B) Quantification of myelinated axon number (left), total axon number (middle), and myelinated axon percentage (right) in the CC from TFEBF/F (Flox), Olig2-Cre; TFEBF/+ (Het) and Olig2-Cre; TFEBF/F (cKO) mice. (C and C’) Representative high-magnification TEM micrographs from P21 Ctl and TFEB cKO CC. (D and E) Quantification of average g-ratios of myelinated axons (E), and as a function of axon diameter (D), in P21 TFEB cKO mice (red) compared with littermate Ctls (TFEBF/F in black and Olig2-Cre; TFEBF/+ in blue). n R 180 axons from 3 animals per genotype. (F and F’) Representative high-magnification TEM micrographs from 6-month-old Ctl and TFEB cKO CC. (G and H) Quantification of average g-ratios of myelinated axons (H), and as a function of axon diameter (G), in 6-month-old TFEB cKO mice (red) as compared with littermate Ctls n R 180 axons from 3 animals per genotype. Error bars represent SEM. Scale bars: 5 mm in (A’) for (A) and (A’); and 0.5 mm in (F’) for (C)–(F’). *p < 0.05.

Article Snippet: GFP-TFEB expression construct was generated by subcloning mouse TFEB cDNA into a pEGFP-N1-Flag vector, which was a gift from Patrick Calsou (Addgene plasmid, #60360) (Britton et al., 2014).

Techniques:

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis.

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: Figure 6. TFEB Promotes the Expression of Subsets of ER Stress Genes and Induces the Pro-apoptotic Factor PUMA in Pre-OLs (A) Volcano plot showing differentially expressed genes in pre-OLs acutely purified from P12 TFEB cKO (Olig2-Cre; TFEBF/F) compared with those purified from littermate Ctl mice (Olig2-Cre; TFEBF/+). Each dot represents a gene. Blue color dots are genes up- or downregulated more than 2-fold. Four selected genes are denoted and highlighted in red. (B) Heatmap showing expression levels (in z scores) of genes up- or downregulated more than 2-fold in TFEB cKO pre-OLs by RNA sequencing (RNA-seq). Replicates of TFEB heterozygous Ctl cells (Het) and KO cells from distinct clusters are shown by the dendrogram. Each row corresponds to a gene, and rows are ordered by fold change values from the highest to the lowest. See Table S1 for the full gene list. (C) Gene ontology (GO) term analysis (Protein Analysis through Evolutionary Relationships [PANTHER] Overrepresentation Test) by using genes downregulated more than 2-fold as input. The top 10 most enriched biological process (BP) terms are shown. Only the narrowest term from each hierarchical group in the analysis is plotted. The full list of enriched terms can be found in Table S2. (D) qRT-PCR of PUMA(Bbc3) mRNA expression in differentiating OLs from TFEB cKO mice and littermate Ctls. n = 3 biological replicates for both genotypes and for each time points. Error bars represent SEM. *p < 0.05.

Article Snippet: GFP-TFEB expression construct was generated by subcloning mouse TFEB cDNA into a pEGFP-N1-Flag vector, which was a gift from Patrick Calsou (Addgene plasmid, #60360) (Britton et al., 2014).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a ) The mRNA levels of SYT11 and GAPDH in Control and ATP13A2-knockdown HeLa cells were measured by real-time PCR. The experiment (with triplicates) was repeated an additional two times and all were similarly significant. ( b ) Primary cultures of mouse neurons transduced with Control or ATP13A2 lentiviral shRNAs for 5 days were used to measure the mRNA levels of SYT11 and GAPDH by real-time PCR. The graph shows the mean±s.d. of four independent experiments. * P <0.05 (two-tailed paired Student's t -test). ( c ) Primary cultures of mouse neurons transduced with Control or ATP13A2 lentiviral shRNAs (#A and #B) were lysed and blotted against SYT11 and actin. A representative experiment with triplicates is shown. ( d ) The mRNA levels of SYT11 and GAPDH in HeLa cells transfected with empty pcDNA3.1 or ATP13A2 WT were measured by real-time PCR. A representative experiment (with triplicates) of three similarly significant independent experiments is shown. ( e ) HeLa cells transfected with empty pcDNA3.1, ATP13A2 WT, ATP13A2 delC or ATP13A2 i16 mutants were used to measure the mRNA levels of SYT11 and GAPDH by real-time PCR. The experiment was repeated and both experiments were similarly significant. ( f ) HeLa cells transfected with V5-tagged ATP13A2 WT, ATP13A2 delC or ATP13A2 i16 constructs were immunostained against V5 and LAMP1 and imaged using confocal microscopy (scale bar 10 μM). Unless otherwise stated, all the graphs represent mean±s.d. and statistical significance was determined using two-tailed unpaired Student's t -test. ** P <0.01; *** P <0.001; NS, not significant.

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Real-time Polymerase Chain Reaction, Transduction, Two Tailed Test, Transfection, Construct, Confocal Microscopy

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a , b ) Control and ATP13A2-knockdown HeLa cells, treated with 300 nM torin-1 (or DMSO) for 4 h ( a ), and HeLa cells transfected with empty vector or ATP13A2 WT ( b ) were used for cytosolic and nuclear fractionation. The different fractions were blotted for TFEB, Lamin-B and GAPDH. ( c ) ATP13A2-knockdown cells or ATP13A2-overexpressing cells (and respective controls) growing in coverslips were fixed with cold methanol and immunostained against TFEB. TFEB translocation to the nucleus was assessed by DAPI co-localization. Cells were imaged by confocal microscopy (scale bar, 15 μM). ( d , e ) Primary cultures of mouse neurons were transduced with Control or ATP13A2 lentiviral shRNAs for 5 days and subsequently used for cytosolic/nuclear fractionation where the different fractions were blotted for TFEB, Lamin-B and GAPDH ( d ). Cells seeded in coverslips were also used for TFEB immunostaining ( e ) (scale bar, 10 μM). ( f , g ) Control and TFEB-knockdown (transfected with pool or deconvoluted oligos) HeLa cells, transfected with empty vector or ATP13A2 WT for the last 24 h ( f ), or Control and ATP13A2-knockdown HeLa cells transfected with empty pCMV, TFEB WT or TFEB S142A for the last 24 h ( g ), were used to measure the mRNA levels of SYT11 and GAPDH by real-time PCR. Representative experiments of two independent experiments with triplicates are shown. ( h ) Control and ATP13A2 WT-overexpressing HeLa cells were used to perform chromatin immunoprecipiation (ChIP). Two different antibodies were tested for TFEB-immunoprecipitation and two pairs of primers designed against the putative TFEB-binding site 2 on the promoter of SYT11 were used for qPCR. The TFEB binding to SYT11 promoter is represented. The experiment (with triplicates) was repeated and both experiments were similarly significant. ( i ) HeLa cells were transfected with empty pcDNA3.1 or ATP13A2 WT simultaneously with WT or CLEAR-site 2 mutant SYT11 promoter-GLuc/SEAP. Medium was collected 48 h later. Secreted Gaussia luciferase and SEAP were measured. A representative experiment (with triplicates) of two independent experiments is shown. All the graphs represent mean±s.d. and statistical significance was determined using two-tailed unpaired Student's t -test. * P <0.05, ** P <0.01; # P <0.05, ## P <0.01. CF, cytosolic fraction; NF, nuclear fraction.

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Transfection, Plasmid Preparation, Fractionation, Translocation Assay, Confocal Microscopy, Transduction, Immunostaining, Real-time Polymerase Chain Reaction, Immunoprecipitation, Binding Assay, Mutagenesis, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a ) Control and ATP13A2-knockdown HeLa cells growing in coverslips were fixed and immunostained against LAMP1 and phospho-mTOR. Cells were imaged by confocal microscopy (scale bar, 15 μM). Pearson's co-localization coefficient and fraction of phospho-mTOR-positive lysosomes were determined using ImageJ. The plotted data are means±s.d. n =20 cells. * P <0.05; ## P <0.01 (two-tailed unpaired Student's t -test). The experiment was repeated an additional two times. ( b ) ATP13A2-knockdown and ATP13A2-overexpressing HeLa cells (for 96 and 24 h, respectively) were lysed and blotted for phospho-p70S6K, total-p70S6K and actin. ( c ) Primary cultures of mouse neurons were transduced with Control or ATP13A2 lentiviral shRNAs for 5 days and used for western blotting against phospho-p70S6K, total-p70S6K and actin. ( d ) HeLa cells transfected with empty vector, ATP13A2 WT, ATP13A2 delC mutant or ATP13A2 i16 mutant for 24 h were lysed and blotted against phospho-p70S6K, total-p70S6K and actin. ( e ) Control and ATP13A2-knockdown HeLa cells were treated with 100 nM rapamycin for 20 h. The mRNA levels of SYT11 and GAPDH were measured by real-time PCR. A representative experiment (with triplicates) of two independent experiments is shown. The graph represents mean±s.d. ** P <0.01; # P <0.05 (two-tailed unpaired Student's t -test).

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Confocal Microscopy, Two Tailed Test, Transduction, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a ) Control and SYT11-knockdown HeLa cells were transfected with GFP-LC3 vector. Cells in coverslips were imaged by fluorescence microscopy (scale bar, 10 μM) and GFP-LC3 dots were quantified using ImageJ. The quantification shows the mean±s.d. of a minimum of 200 cells per replicate (in a total of three replicates). * P <0.05 (two-tailed unpaired Student's t -test). ( b , c ) Control and SYT11-knockdown (transfected with pool or deconvoluted oligos) HeLa cells were treated with 200 nM bafilomycin A1 (BAF A1) for the last 12 h in full medium ( b ) or with 400 nM BAF A1 for the last 4 h in HBSS medium ( c ). Cells were lysed and blotted for LC3 and actin. ( d , e ) Control and SYT11-knockdown SK-N-SH cells ( d ) or primary cultures of mouse neurons transduced with Control or SYT11 lentiviral shRNAs ( e ) were treated with 200 nM bafilomycin A1 (BAF A1) for the last 12 h. Lysates were blotted for LC3 and actin. ( f ) GFP-P62 HEK293 Flp-In T-REx cells were transfected with two rounds of Control or SYT11 siRNA. After the second round of transfection, GFP-P62 expression was induced by tetracycline (1 μg ml −1 ) for 24 h. Cells were then rinsed twice with PBS and incubated with normal cell culture medium (to stop transgene expression) for 18 h. Lysates were blotted for GFP and actin. The graph shows mean±s.d. of five independent experiments. * P <0.05 (two-tailed paired Student's t -test). ( g , h ) Control and SYT11-knockdown HeLa cells stably expressing tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) were fixed with 2% paraformaldehyde for 4 min and imaged by confocal microscopy (scale bar, 15 μM) ( g ) or analysed on an automated ArrayScan system ( h ). Means±s.e.m. of number of autophagosomes (AP) and autolysosomes (AL) per cell and area of green and red vesicles are shown in the graphs. BAF A1-treated cells (400 nM for 4 h) were used as a control. Approximately 2,000 cells were analysed per condition in each Cellomics experiment and the experiment was repeated three additional times. * P <0.05; *** P <0.001 (two-tailed paired Student's t -test).

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Two Tailed Test, Transduction, Expressing, Incubation, Cell Culture, Stable Transfection, Confocal Microscopy

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a , b ) Control and SYT11-knockdown HeLa cells growing in coverslips were immunostained for LAMP1 and LC3 ( a ) or only LAMP1 ( b ) and imaged by confocal microscopy (scale bar, 15 μM ( a ) or 10 μM ( b )). Pearson's co-localization coefficient and the fraction of LAMP1-positive LC3 vesicles were determined using ImageJ. The plotted data are means±s.d. of at least 20 cells ( a ). The experiments were repeated an additional two times. ( c ) Control and SYT11-knockdown HeLa cells were loaded with LysoSensor yellow/blue and analysed by live imaging (scale bar, 5 μM). The graph shows the mean±s.e.m. of the yellow/blue intensity ratio of images obtained from 10 fields. A representative experiment of two independent experiments is shown. ( d ) Control and SYT11-knockdown HeLa cells were used to measure cathepsin-L activity in vitro by incubating cell lysates with 200 μM Ac-FR-AFC for 2 h at 37 °C. The graph represents fluorescence means±s.d. of three independent experiments with triplicates each condition. *** P <0.001 (two-tailed paired Student's t -test). ( e ) Lysates of control and SYT11-knockdown HeLa cells were blotted against Cathepsin-L and actin. The Cathepsin-L antibody detects the pro-form, the intermediate and the mature/processed forms of the protein. ( f , g ) Control and ATP13A2-knockdown HeLa cells ( f ) or primary cultures of mouse neurons transduced with Control or ATP13A2 lentiviral shRNAs ( g ) were treated with 200 nM BAF A1 for the last 12 h. Cells were lysed and blotted against LC3 and actin. ( h ) Fixed control and ATP13A2-knockdown HeLa cells were immunostained for LAMP1 and imaged by confocal microscopy (scale bar, 15 μM). ( i ) Control and ATP13A2-knockdown HeLa cells were loaded with LysoSensor yellow/blue and analysed by live imaging (scale bar, 5 μM). The graph shows the mean±s.e.m. of the yellow/blue intensity ratio of images obtained from 10 fields. A representative experiment of two independent experiments is shown. Unless otherwise stated, all the graphs represent mean±s.d. and statistical significance was determined using two-tailed unpaired Student's t -test. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Confocal Microscopy, Imaging, Activity Assay, In Vitro, Fluorescence, Two Tailed Test, Transduction

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a ) HeLa cells were transfected with Control, SYT11 siRNA, ATP13A2 siRNA or SYT11 siRNA in combination with ATP13A2 siRNA. Cells were treated with 200 nM bafilomycin A1 (BAF A1) for the last 12 h and lysates were blotted for LC3 and actin. ( b , c ) Control and ATP13A2-knockdown HeLa cells were transfected with empty pEGFP or pEGFP-SYT11 in the last 30 h of the experiment ( b , c ). Cells were treated with 200 nM BAF A1 for the last 12 h ( c ). Cell lysates were blotted for LC3, GFP (SYT11) and actin. ( d , e ) HeLa WT ( d ) or ATG16L CRISPR ( e ) cells were transfected with Control or ATP13A2 siRNA for 5 days. In the last 48 h, cells were transfected with empty vector and pEGFP-SYT11 for 5 h, followed by transfection with empty pEGFP+GFP-α-synuclein A53T. Cells were lysed and blotted for GFP. Representative experiments with triplicates of three independent experiments are shown. Levels of α-synuclein A53T are expressed as a ratio to GFP. ( f ) Lysates obtained from ATG16L-knockout cells produced by CRISPR/Cas9 editing and control HeLa cells were blotted against ATG16L, LC3 and actin in order to validate the knockout efficiency and autophagy competence. ( g ) Control and ATP13A2-knockdown HeLa cells were transfected with empty pcDNA3.1- myc /His or pcDNA3.1-SYT11- myc /His in the last 30 h of the experiment. Cells were subsequently loaded with LysoSensor Yellow/Blue and analysed by live imaging (scale bar, 10 μM). The graph shows the mean±s.e.m. of the yellow/blue intensity ratio of images obtained from 10 fields. ( h ) HeLa cells were transfected with Control or ATP13A2 siRNA for 5 days. In the last 48 h, cells were transfected with empty pCMV, pCMV TFEB WT or TFEB S142A for 5 h, followed by transfection with empty pEGFP+GFP-α-synuclein A53T. Cells were lysed and blotted for GFP. A representative experiment with triplicates of two independent experiments is shown. Levels of α-synuclein A53T are expressed as a ratio to GFP. All the graphs represent mean±s.d. and statistical significance was determined using two-tailed unpaired Student's t -test. * P <0.05; ** P <0.01; *** P <0.001; # P <0.05; ### P <0.001; NS, not significant.

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Transfection, CRISPR, Plasmid Preparation, Knock-Out, Produced, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: ( a ) Control and ATP13A2-knockdown HeLa cells were transfected with pEGFP-SYT11 for the last 24 h. Cell lysates were blotted against GFP and actin. ( b ) HeLa cells were transfected with empty vector or ATP13A2 WT simultaneously with pEGFP-SYT11 for 24 h. Cell lysates were blotted against GFP and actin. ( c ) Control and ATP13A2-knockdown HeLa cells were transfected with GFP-SYT11 for the last 24 h. In the last 4 h of the experiment, cells were treated with 50 μg ml −1 cycloheximide for the indicated time points. Cell lysates were blotted against GFP and actin. Densitometric quantification of the bands was performed and the normalized data (GFP-SYT11 levels to actin levels) of three independent experiments is plotted in the graph. ( d ) HeLa cells were transfected with empty vector or ATP13A2 WT simultaneously with HA-Ubiquitin and GFP-SYT11 for 24 h. Cell lysates were subsequently used for GFP-SYT11 immunoprecipitation and western blotting against GFP, ubiquitin and actin. ( e ) Control and ATP13A2-knockdown HeLa cells were transfected with HA-Ubiquitin and GFP-SYT11 for the last 24 h. Lysates were used for GFP-SYT11 immunoprecipitation and subsequent western blotting against ubiquitin (P4D1 antibody), K48-linkage-specific polyubiquitin conjugates, GFP and actin. ( f ) Control and ATP13A2-knockdown HeLa cells were transfected with GFP-SYT11 for the last 24 h. In the last 5 h, cells were pre-incubated with 10 μM MG132 (or DMSO) and further treated with 50 μg ml −1 cycloheximide for the indicated time points. Cell lysates were blotted against GFP and actin. Densitometric quantification of the bands was performed and the normalized data (GFP-SYT11 levels to actin levels) of three independent experiments is plotted in the graph. All the graphs represent mean±s.d. and statistical significance was determined using two-tailed paired Student's t -test. * P <0.05; ** P <0.01.

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

doi: 10.1038/ncomms11803

Figure Lengend Snippet: Orange arrows identify the sequence of events that occur when ATP13A2 is depleted from cells, which in turn leads to depletion of SYT11, while green edges identify the sequence of events when ATP13A2 and SYT11 remain under steady-state conditions. Overall, we propose that ATP13A2 depletion decreases TSC2 levels, by a mechanism dependent on MYCBP2-induced ubiquitination, which in turn induces activation of mTORC1 and decreased TFEB-mediated transcription of SYT11 . In parallel, ATP13A2 depletion induces SYT11 ubiquitination and degradation. Both events contribute to a decrease of SYT11 levels, which induces lysosomal dysfunction, autophagy blockage and increased accumulation of α-synuclein A53T.

Article Snippet: The following constructs were also used in this work: empty pEFGP; pEGFP-LC3; pEGFP-SYT11 (kindly provided by Dr Stefan Pulst) ; pCMV6-SYT11-TurboGFP (RG205445, Origene); pcDNA3.1- myc -His (Invitrogen); pcDNA3.1-SYT11/ myc -His; SYT11 promoter-GLuc/SEAP pEWZX-PG04 (HPRM12158-PG04, GeneCopoeia); pEGFP-α-synuclein A53T; pcDNA3.1; pcDNA3.1 ATP13A2 WT, pcDNA3.1 ATP13A2 delC (3057 delC) mutant and pcDNA3.1 ATP13A2 i16 (1632_1653dup22) mutant (all V5-tagged) (kindly provided by Dr Christian Kubisch) ; empty pCMV; pCMV TFEB WT-3xFlag and pCMV TFEB S142A-3xFlag constitutively active mutant (kindly provided by Dr Andrea Ballabio) ; pcDNA3 Flag-TSC2 (#14129, Addgene) ; pcDNA3.1 HA-ubiquitin; pSpCas9n(BB)-2A-GFP (PX461) vector (#48410, Addgene).

Techniques: Sequencing, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: Bafilomycin A1 (Baf) increases TDP-43, TSG101, and LC3-II levels in the EV fraction. A – C , HeLa cells expressing mRFP-GFP-LC3 were exposed to vehicle (DMSO), 100 nM Baf, 1 μM MG, or 100 nM Vac for 24 h. A , representative images of these cells are shown. Nuclei were stained with DAPI ( blue ). Scale bar represents 20 μm. B , autophagosome and ( C ) autolysosome numbers calculated from three independent experiments including at least 20 cells are shown. D and E , HeLa cells exposed to vehicle (DMSO), 100 nM Baf, 1 μM MG, or 100 nM Vac for 24 h were stained with AcidiFluor ORANGE. D , representative images of live cells stained with AcidiFluor ORANGE ( red ) in each treatment. Nuclei were stained with Hoechst 33342 ( blue ). Scale bar represents 20 μm. E , relative signal intensities normalized against cells exposed to vehicle were calculated from three independent experiments including at least 330 cells. F – J , HeLa cells were exposed to DMSO, 100 nM Baf, 1 μM MG, or 100 nM Vac for 24 h. The cell, P1 (20,000 g pellet), and P2 (110,000 g pellet) fractions were prepared as shown in the section. F , representative immunoblots (TDP-43 detected by monoclonal, C-terminal, and N-terminal antibodies, TSG101, LC3, and α-tubulin) of each fraction are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. G – J , densitometric data on ( G ) full-length TDP-43, ( H ) cleaved TDP-43, ( I ) TSG101, and ( J ) LC3-II in the cell, P1, and P2 fractions were calculated from immunoblotting results. Relative signal intensities normalized against cells exposed to Baf are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test or Mann–Whitney U test. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EV, extracellular vesicle; LC3, microtubule-associated proteins 1A/1B light chain 3B; MG, MG132; mRFP, monomeric red fluorescent protein; TDP-43, TAR DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein; Vac, vacuolin-1.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Expressing, Staining, Western Blot, Two Tailed Test, MANN-WHITNEY, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: Inhibition of autophagosome–lysosome fusion by bafilomycin A1 (Baf) is required for TDP-43 secretion via autophagy-associated ve sicles. HeLa cells were exposed to indicated treatment for 24 h. A , representative immunoblots (TDP-43 detected by the monoclonal antibody, TSG101, LC3, and α-tubulin) of each fraction are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. B – E , densitometric data on ( B ) full-length TDP-43, ( C ) cleaved TDP-43, ( D ) TSG101, and ( E ) LC3-II in the cell, P1, and P2 fractions were calculated from immunoblotting results. Relative signal intensities normalized against cells exposed to Baf are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. LC3, microtubule-associated proteins 1A/1B light chain 3B; Rapa, rapamaycin; SS, serum starvation; TDP-43, TAR DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Inhibition, Western Blot, Two Tailed Test, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: TARDBP knockdown enhances the extracellular release of LC3-II from bafilomycin A1 (Baf)–treated cells. HeLa cells transfected with control or TARDBP siRNA for 48 h were further cultured in 100 nM Baf-containing medium for 24 h. The cell, P1, and P2 fractions were prepared as shown in the section. A , representative immunoblots (TDP-43, TSG101, LC3, and α-tubulin) are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. B – E , densitometric data on ( B ) full-length TDP-43, ( C ) cleaved TDP-43, ( D ) TSG101, and ( E ) LC3-II in each fraction were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with control siRNA are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, TAR DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Transfection, Cell Culture, Western Blot, Two Tailed Test, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: The C-terminal region of TDP-43 is required for bafilomycin A1 (Baf)-induced TDP-43 secretion. A and B , HeLa cells stably expressing mCherry-LC3 transfected with GFP-tagged full-length (amino acids 1–414), the N-terminal fragment (amino acids 1–273), or C-terminal fragment (amino acids 162–414) of TDP-43 were exposed to 100 nM Baf for 24 h. A , representative confocal images of live cells in each treatment are shown. Nuclei were stained with Hoechst 33342. Scale bar represents 20 μm. Arrowheads indicate the colocalization of TDP-43 amino acids 162 to 414 and mCherry-LC3. B , densitometric data of fluorescence images were calculated from three independent experiments including at least 20 cells. The percentage of the GFP-TDP-43 signal colocalized with mCherry-LC3 to the total GFP-TDP-43 signal is shown. C – E , HeLa cells transfected with TDP-43 amino acids 1 to 414, TDP-43 amino acids 1 to 273, or TDP-43 amino acids 162 to 414 were exposed to 100 nM Baf for 24 h, and the cell, P1, and P2 fractions were prepared. C , representative immunoblots (TDP-43, GFP, and α-tubulin) of the cell, P1, and P2 fractions are shown. D and E , the ratio of GFP-TDP-43 levels in the ( D ) P1 and ( E ) P2 fractions to the cellular fraction calculated from the densitometric data of immunoblotting results are normalized against cells transfected with TDP-43 amino acids 162 to 414. In bar graphs, data are presented as means ± SD (N = 3). Values with a different superscript indicate p < 0.05 by Tukey’s post hoc test. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, TAR DNA-binding protein 43 kDa.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Fluorescence, Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: The bafilomycin A1 (Baf) treatment induces the localization of GFP-tagged C-terminal TDP-43 (amino acids 162–414) and mCherry-LC3 to the plasma membrane. HeLa cells transfected with the TDP-43 amino acids 162 to 414 construct or mCherry-LC3 construct were exposed to vehicle or 100 nM Baf for 24 h. A and B , representative confocal images of live cells transfected with the TDP-43 amino acids 162 to 414 construct ( green ) exposed to ( A ) vehicle or ( B ) 100 nM Baf are shown. The plasma membrane (PM) was stained with PlasMem Bright Red ( red ). Nuclei were stained with Hoechst 33342. C and D , representative confocal images of live cells transfected with the mCherry-LC3 construct ( red ) exposed to ( C ) vehicle or ( D ) 100 nM Baf are shown. The PM was stained with PlasMem Bright Green ( green ). The image surrounded by a yellow square was magnified. Scale bar represents 5 μm. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, TAR DNA-binding protein 43 kDa.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Membrane, Transfection, Construct, Staining, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: ATG16L1 is required for bafilomycin A1 (Baf)-induced TDP-43 secretion. A and B , ATG16L1 WT or KO HeLa cells transfected with the mCherry-LC3 construct were exposed to 100 nM Baf for 24 h. Representative confocal live-cell images of ATG16L1 ( A ) WT or ( B ) KO cells transfected with the mCherry-LC3 ( red ) construct exposed to Baf. The plasma membrane (PM) was stained with PlasMem Bright Green ( green ). The image surrounded by a yellow square was magnified. Scale bar represents 5 μm. C – F , ATG16L1 WT or KO HeLa cells were exposed to 100 nM Baf for 24 h. The cell, P1, and P2 fractions were prepared as shown in the section. C , representative immunoblots (ATG16L1, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. D – F , densitometric data on ( D ) full-length TDP-43, ( E ) cleaved TDP-43, and ( F ) TSG101 in the cell, P1, and P2 fractions were calculated from immunoblotting results. Relative signal intensities normalized against ATG16L1 WT cells are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Transfection, Construct, Membrane, Staining, Western Blot, Two Tailed Test, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: Chloroquine, an inhibitor of autolysosome formation and lysosomal acidification, does not increase the secretion of TDP-43 or LC3-II. A and B , HeLa cells stained with DALGreen were exposed to vehicle (DMSO), 100 nM Baf, 100 nM Con, or 100 μM CQ for 2 h. A , representative images of live cells stained with DALGreen ( green ) in each treatment are shown. Nuclei were stained with Hoechst 33342 ( blue ). Scale bar represents 50 μm. B , relative signal intensities normalized against cells exposed to vehicle were calculated from three independent experiments including at least 74 cells. C and D , HeLa cells exposed to vehicle (DMSO), 100 nM Baf, 100 nM Con, or 100 μM CQ for 24 h were stained with AcidiFluor ORANGE. C , representative images of live cells stained with AcidiFluor ORANGE ( red ) in each treatment. Nuclei were stained with Hoechst 33342 ( blue ). Scale bar represents 20 μm. D , relative signal intensities normalized against cells exposed to vehicle were calculated from three independent experiments including at least 332 cells. E – I , HeLa cells were exposed to vehicle (DMSO), 100 nM Baf, 100 nM Con, or 100 μM CQ for 24 h. The cell, P1, and P2 fractions were prepared as shown in the section. E , representative immunoblots (TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. F – I , densitometric data on ( F ) full-length TDP-43, ( G ) cleaved TDP-43, ( H ) TSG101, and ( I ) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against cells exposed to the vehicle are shown. In bar graphs, data are presented as means ± SD (N = 3). Values with a different superscript indicate p < 0.05 by Tukey’s post hoc test. Baf, bafilomycin A1; Con, concanamycin A; CQ, chloroquine; DMSO, dimethyl sulfoxide; LC3-II, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Staining, Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: The knockdown of GRN increases the secretion of TDP-43 and LC3-II in an autophagy-dependent manner. A and B , HeLa cells transfected with control or GRN siRNA for 48 h were stained with DALGreen and incubated for 2 h. A , representative live-cell images stained with DALGreen ( green ) are shown. Nuclei were stained with Hoechst 33342 ( blue ). Scale bar represents 50 μm. B , relative signal intensities normalized against cells transfected with control siRNA were calculated from three independent experiments including at least 78 cells. C and D , HeLa cells transfected with control or GRN siRNA for 48 h were stained with AcidiFluor ORANGE, and the acidity of lysosomes was compared. C , representative live-cell images stained with AcidiFluor ORANGE and photographed at a bright field are shown. Scale bar represents 100 μm. D , relative signal intensities normalized against cells transfected with control siRNA were calculated from three independent experiments including at least 627 cells. E – I , HeLa cells transfected with control or GRN siRNA were incubated for 72 h. The cell, P1, and P2 fractions were prepared as shown in the section. E , representative immunoblots (PGRN, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. F – I , densitometric data on ( F ) full-length TDP-43, ( G ) cleaved TDP-43, ( H ) TSG101, and ( I ) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with GRN siRNA are shown. J – M , ATG16L1 KO HeLa cells transfected with control or GRN siRNA were incubated for 72 h. The cell, P1, and P2 fractions were prepared as shown in the section. N is a sample from normal HeLa cells. J, representative immunoblots (PGRN, ATG16L1, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. K – M , densitometric data on ( K ) full-length TDP-43, ( L ) cleaved TDP-43, and ( M ) TSG101 were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with GRN siRNA are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test or Mann–Whitney U test. LC3, microtubule-associated proteins 1A/1B light chain 3B; PGRN, progranulin; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Transfection, Staining, Incubation, Western Blot, Two Tailed Test, MANN-WHITNEY, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: The KO of Grn increases the secretion of TDP-43 and LC3-II from MEFs. A , the base sequences of the genome-edited regions of PGRN in WT and KO mice were elucidated by Sanger sequencing. Arrowheads in the WT alignment show the two bases deleted by CRISPR–Cas9 genome editing. B , representative confocal images of PGRN ( green ) immunostaining of MEFs derived from PGRN WT and KO mice. Nuclei were stained with DAPI ( blue ). Arrowheads indicate vesicle-like structures. The image surrounded by a white square in WT MEFs was magnified. Scale bar represents 5 μm. C – H , PGRN WT and KO MEFs were cultured for 72 h. The cell, P1, and P2 fractions were prepared as shown in the section. C , representative immunoblots (PGRN, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. D , the P1 and P2 fractions treated with or without PNGase F were immunoblotted with PGRN and α-tubulin antibodies. Representative immunoblots of the cell, P1, and P2 fractions are shown (N = 3). E – H , densitometric data on ( E ) full-length TDP-43, ( F ) cleaved TDP-43, ( G ) TSG101, and ( H ) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against PGRN KO MEFs are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. DAPI, 4′,6-diamidino-2-phenylindole; LC3-II, microtubule-associated proteins 1A/1B light chain 3B; MEF, mouse embryonic fibroblast; PGRN, progranulin; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Sequencing, CRISPR, Immunostaining, Derivative Assay, Staining, Cell Culture, Western Blot, Two Tailed Test, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: Treatments leading to the secretion of TDP-43 promote TFEB-GFP nuclear localization in SH-SY5Y cells. A and B , SH-SY5Y cells stably expressing TFEB-GFP were exposed to DMSO, 100 nM Baf, 100 nM Con, 100 μM CQ, or 100 nM Vac for 24 h. A , representative confocal images are shown. Nuclei were stained with DAPI ( blue ). Scale bar represents 20 μm. B , the percentage of cells in which TFEB-GFP translocated in the nucleus calculated from three independent experiments including at least 102 cells is shown. C and D , SH-SY5Y cells stably expressing TFEB-GFP were transfected with control, TARDBP #1, TARDBP #2, GRN , or STX17 siRNA for 48 h. C , representative confocal images are shown. Nuclei were stained with DAPI ( blue ). Scale bar represents 20 μm. D , the percentage of cells in which TFEB-GFP translocated in the nucleus calculated from three independent experiments including at least 120 cells is shown. E – I , SH-SY5Y cells transfected with control or TFEB siRNA were incubated for 72 h. The cell, P1, and P2 fractions were prepared as shown in the section. E , representative immunoblots (TFEB, TDP-43, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. F – I , densitometric data on ( F ) TFEB, ( G ) full-length TDP-43, ( H ) cleaved TDP-43, and ( I ) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with control siRNA are shown. In bar graphs, data are presented as means ± SD (N = 3). Values with a different superscript indicate p < 0.05 by Tukey’s post hoc test. ∗ indicates p < 0.05 by the two-tailed unpaired t test. Baf, bafilomycin A1; Con, concanamycin A; CQ, chloroquine; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; TDP-43, transactive response DNA-binding protein 43 kDa; TFEB, transcription factor EB; Vac, vacuolin-1.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520 ), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505 ), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2-AP, RRID: AB_615042 ), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161 ), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104 ; catalog no.: 14497-1-AP, RRID: AB_2208090 ), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1-AP, RRID: AB_10642150 ), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291 ), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794 ) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70-102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit anti-Syntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757 ) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151 ) from BD Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504 ) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951 ) from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody (catalog no.: BA-2000, RRID: AB_2313581 ), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606 ), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217 ) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996 ), and Alexa Fluor 488–conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320 ) from Abcam.

Techniques: Stable Transfection, Expressing, Staining, Transfection, Incubation, Western Blot, Two Tailed Test, Binding Assay