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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Control, Knockdown, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Control, Knockdown, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Control, Knockdown, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Control, Knockdown, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Control, Knockdown, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Transfection, Control, Knockdown, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Control, Knockdown, Transfection, Plasmid Preparation, Ubiquitin Proteomics, 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: Mutagenesis of the human WT SYT11 promoter-GLuc-SEAP vector (GeneCopoeia) (CLEAR-Site 2 in SYT11 promoter; CTCAAGTGAT>CAAAAGAAAT) was generated with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Stratagene), according to manufacturer's instructions, using the set of primers listed in .

Techniques: Sequencing, Ubiquitin Proteomics, Activation Assay

Journal: Frontiers in Transplantation

Article Title: Mechanisms inducing differentiation of adult islet progenitor-like cells into functional islet-like organoids

doi: 10.3389/frtra.2026.1740314

Figure Lengend Snippet: Requirement of CN/NFAT signaling to recruit p300 to RFX6 and NEUROD1 promoters during ISX9-induced IPC islet organoid differentiation (A) induction of NFAT family isoform genes in IPC clusters treated with ISX9 for 24 h in the presence of CN inhibitor FK506. (B) RFX6 and NEUROD1 promoter activation by ISX9 in IPCs in the presence of FK506 or transfected with gene vectors overexpressing dominant negative NFAT (dnNFAT) and mutated control (dnNFATm). (C) ChIP assay of association of NFATC2, p300, HDAC1, HDAC2, and HDAC3 with RFX6, NEUROD1, and NEUROG3 promoters upon 6 h treatment of IPCs with ISX9 with and without 24 h pretreatment with ITF2357. Graphed values are expressed as mean ± SD. Asterisks above bars indicate statistically significant differences (* p < 0.05, *** p < 0.001) in mean values for treatments based on a two-way ANOVA and Sidak's multiple comparison test. Data shown are results from at least three independent experiments using IPCs derived from three individual donors.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Control, Comparison, Derivative Assay

Journal: Frontiers in Transplantation

Article Title: Mechanisms inducing differentiation of adult islet progenitor-like cells into functional islet-like organoids

doi: 10.3389/frtra.2026.1740314

Figure Lengend Snippet: Schematic overview of IPC isolation, expansion, and differentiation. Adult human pancreatic tissue obtained from islet cell isolation fractions was expanded in vitro to generate a CD9 + PROCR + IPC-enriched population. IPCs were identified using transcriptomic and phenotypic analyses (scRNA-seq, flow cytometry, and immunofluorescence). An RGS16 + organoid-forming subset was characterized within the expanded population. IPC clusters were subsequently subjected to ISX9-mediated differentiation. ISX9 treatment stimulated calcineurin (CN)/NFAT signaling, promoted NFATC2–p300 association and binding at RFX6 and NEUROD1 promoters, and induced downstream endocrine transcriptional programs (NGN3, RFX6, NEUROD1, NKX2.2, NKX6.1, MAFA), resulting in functional islet organoids.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Isolation, Cell Isolation, In Vitro, Flow Cytometry, Immunofluorescence, Binding Assay, Functional Assay