Journal: bioRxiv

Article Title: TBC1D15 potentiates lysosomal regeneration from damaged membranes

doi: 10.1101/2022.12.14.520480

Figure Lengend Snippet: a) HeLa cells were treated with control, TFEB or TFE3 siRNA for 48 h. Lysosomal damage was induced by 1mM LLOMe for 2 h. The LLOMe containing media was then replaced with fresh media to allow recovery. Cells were fixed at different time points after LLOMe washout. LysoTracker Red was loaded 30 min before the cells were fixed for confocal imaging. LysoTracker Red intensity was measured at different time points before and after LLOMe washout. Data are means ± SEM of 60 cells taken from three independent experiments. b) Schematic of the RFP GFP dual-tagged Gal3 (tfGal3) reporter construct used for the lysosomal regeneration flux assay. c) HeLa cells with stable expression of tfGal3 were treated with control or TFEB siRNA for 48 h. Lysosomal damage was induced by treating cells with LLOMe (1mM, 2 h). Cells were cultured in LLOMe-free medium for different durations similar to panel (a). The number of red Galectin3 puncta was counted at each time point before and after LLOMe washout. TFEB knockdown efficiency is noted by western blotting. d) Graph showing numbers of red Galectin3 puncta per cell obtained from the experiments in panel (c). Data are means ± SEM of 50 cells taken from three independent experiments. e) Schematic of the LAMP1-APEX proximity labelling assay. f) Volcano plot to compare the biotinylated landscape of APEX2-tagged LAMP1 with and without treatment with 1mM LLOMe for 2 h. Green and yellow circles indicate biotinylated proteins that are significantly enriched and significantly depleted in LLOMe-treated cells, respectively. g) Major categories and candidate proteins enriched by LLOMe treatment that are relevant to this study following pathway analysis in Perseus.

Article Snippet: The siRNAs for TBC1D15 (sc-95660), TFEB (sc-38509), TFE3 (sc-38507), ATG3 (sc-72582), ATG12 (sc-72578), ATG13 (sc-97013), ATG16L (sc-72580), Rubicon (sc-78326), DNM2 (sc-35236), LIMP2 (sc-41546) and TMEM192 (sc-89327) were purchased from Santa Cruz Biotechnology.

Techniques: Control, Imaging, Construct, Flux Assay, Expressing, Cell Culture, Knockdown, Western Blot

Journal: bioRxiv

Article Title: TBC1D15 potentiates lysosomal regeneration from damaged membranes

doi: 10.1101/2022.12.14.520480

Figure Lengend Snippet: Role of TFEB, TFE3, and mTOR in lysosomal regeneration following LLOMe-mediated damage. a) HeLa cells were treated with siRNA to deplete endogenous TFEB/TFE3. Lysosomal damage was induced with 1mM LLOMe treatment for 2 h followed by washout for the indicated durations. Cells were loaded with LysoTracker Red for 30 min, fixed, and stained with a TFEB/TFE3 specific antibody. b) HeLa cells were treated with siRNA to deplete endogenous TFEB and LAMP1-RFP was transfected to mark lysosomes. These cells were then subjected to LLOMe treatment (1mM, 2 h) followed by washout for indicated timepoints. DQ-BSA was loaded for the last 30 minutes of different treatment conditions prior to imaging the samples by confocal microscopy. TFEB knockdown efficiency was checked by western blotting c) DQ-BSA signal intensities were compared between control cells and cells depleted for endogenous TFEB as a percentage of the signal observed in control siRNA treated cells without LLOMe. Data are means ± SEM from at least 30 cells taken from three independent experiments. d) mTOR activity was assessed by monitoring the phosphorylation status of p70-S6K by western blotting for indicated treatments. LLOMe:1mM, 2 h, EBSS: 4 h, Torin1: 500nM, 6 h.

Article Snippet: The siRNAs for TBC1D15 (sc-95660), TFEB (sc-38509), TFE3 (sc-38507), ATG3 (sc-72582), ATG12 (sc-72578), ATG13 (sc-97013), ATG16L (sc-72580), Rubicon (sc-78326), DNM2 (sc-35236), LIMP2 (sc-41546) and TMEM192 (sc-89327) were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Transfection, Imaging, Confocal Microscopy, Knockdown, Western Blot, Control, Activity Assay, Phospho-proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: TRPML1 agonist ML-SA5 mitigates uranium-induced nephrotoxicity via promoting lysosomal exocytosis.

doi: 10.1016/j.biopha.2024.117728

Figure Lengend Snippet: Fig. 3. ML-SA5 enhances TFEB nuclear translocation by promoting lysosomal Ca2+ release. For experiment A & B, the mice were treated as described in Fig. 1A. For experiment C & D, the HK-2 cells were exposed to U (300 μM) for 24 h, followed by the treatment with ML-SA5 (10 μM) and/or BAPTA-AM (25 μM) in the complete medium containing 1 mM EGTA for 30 min after washing out the U. A, B Representative immunohistochemical staining images and quantitation of the percentage of PTECs with nuclear TFEB in the S3 segments of the renal PTs in mice with U exposure and ML-SA5 treatment. n = 3 mice per group. C, D Representative immu nofluorescence images and quantitation of nuclear TFEB in HK-2 cells from each experimental group. The result is representative of three independent experiments. All the images share the same scale bars, 20 μm. All data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After culturing for 18 hours, the cells were transfected with TRPML1 (MCOLN1) shRNA (OriGene, USA, #TR303307), TFEB shRNA (Miaolingbio, China, #P31675), or corresponding empty vectors (OriGene, #TR30012; Miaolingbio, #P0684), using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, USA, #L3000015) according to the manufacturer’s instructions.

Techniques: Translocation Assay, Immunohistochemical staining, Staining, Quantitation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: TRPML1 agonist ML-SA5 mitigates uranium-induced nephrotoxicity via promoting lysosomal exocytosis.

doi: 10.1016/j.biopha.2024.117728

Figure Lengend Snippet: Fig. 4. The effects of TRPML1 knockdown on ML-SA5-induced lysosomal exocytosis, intracellular U removal, and U-induced cell damage/death in U-loaded HK-2 cells. TRPML1 shRNA plasmid and corresponding empty vector were utilized to establish TRPML1 knockdown-expressing HK-2 cells and empty vector control cells. The HK-2 cells transfected with TRPML1 shRNA or corresponding empty vector plasmid were exposed to U (300 μM) for 24 h and subsequently treated with ML-SA5 (10 μM) for 30 min. A, B Representative Western blot bands and quantification of TRPML1 in HK-2 cells transfected with empty vector or TRPML1 shRNA plasmid. C The intracellular U content of each experimental group as indicated in the figure. D, E Representative immunofluorescence images and quantification of LAMP-1 protein level on the PM of each experimental group as indicated in the figure. F, G Representative immunofluorescence images and quantification of KIM-1 pro tein level of each experimental group as indicated in the figure. H, I Representative images of Calcein-AM/PI staining and quantification of cell mortality of each experimental group as indicated in the figure. These results are representative of three independent experiments. All the images share the same scale bars, 20 μm. All data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. PM: plasma membrane.

Article Snippet: After culturing for 18 hours, the cells were transfected with TRPML1 (MCOLN1) shRNA (OriGene, USA, #TR303307), TFEB shRNA (Miaolingbio, China, #P31675), or corresponding empty vectors (OriGene, #TR30012; Miaolingbio, #P0684), using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, USA, #L3000015) according to the manufacturer’s instructions.

Techniques: Knockdown, shRNA, Plasmid Preparation, Expressing, Control, Transfection, Western Blot, Immunofluorescence, Staining, Clinical Proteomics, Membrane

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: TRPML1 agonist ML-SA5 mitigates uranium-induced nephrotoxicity via promoting lysosomal exocytosis.

doi: 10.1016/j.biopha.2024.117728

Figure Lengend Snippet: Fig. 5. The effects of TFEB knockdown on ML-SA5-induced lysosomal exocytosis, intracellular U removal, and U-induced cell injury/death in U-loaded HK-2 cells. TFEB shRNA plasmid and the corresponding empty vector were used to establish TFEB knockdown-expressing HK-2 cells and empty vector control cells. The HK-2 cells transfected with TFEB shRNA or corresponding empty vector plasmid were exposed to U (300 μM) for 24 h and subsequently treated with ML-SA5 (10 μM) for 30 min. A, B Representative Western blot bands and quantification of TFEB in HK-2 cells transfected by empty vector or TFEB shRNA plasmid. C The intracellular U content of each experimental group as indicated in the figure. D, E Representative immunofluorescence images and quantification of LAMP-1 protein level on the PM of each experimental group as indicated in the figure. F, G Representative immunofluorescence images and quantification of KIM-1 protein level of each experimental group as indicated in the figure. H, I Representative images of Calcein-AM/PI staining and quantification of cell mortality of each experimental group as indicated in the figure. These results are representative of three independent experiments. All the images share the same scale bars, 20 μm. All data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. PM: plasma membrane.

Article Snippet: After culturing for 18 hours, the cells were transfected with TRPML1 (MCOLN1) shRNA (OriGene, USA, #TR303307), TFEB shRNA (Miaolingbio, China, #P31675), or corresponding empty vectors (OriGene, #TR30012; Miaolingbio, #P0684), using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, USA, #L3000015) according to the manufacturer’s instructions.

Techniques: Knockdown, shRNA, Plasmid Preparation, Expressing, Control, Transfection, Western Blot, Immunofluorescence, Staining, Clinical Proteomics, Membrane

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 Death & Disease

Article Title: Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

doi: 10.1038/s41419-019-1931-4

Figure Lengend Snippet: a , b SMCs were cultured in full-serum medium (10% FBS) and treated with trehalose (0–100 mM) for 24 h. Representative immunoblots and summarized data show the effects of trehalose on the protein expression levels of LC3-II and p62/SQSTM1. c – f SMCs treated with or without 100 mM trehalose cultured in full-serum medium or under starvation condition (Starv, 0.1%FBS) for 24 h. Representative immunofluorescence images ( c ) and quantification data ( d ) show the nuclear translocation of TFEB (green). Nuclei were stained with DAPI. e , f Real-time RT-PCR analyses of TFEB, LAMP-2A, LC3 and p62/SQSTM1 mRNA levels. g , h Immunoblots show the expression of TFEB in cytosolic or nuclear extract of SMCs ( n = 4). i SMCs cultured in full-serum medium were treated with or without 100 mM trehalose in the absence or presence of bafilomycin (Baf, 50 nM) for 24 h. Representative immunoblots and summarized data show the protein expression levels of LC3-II. Scale bar = 50 µm. * P < 0.05 ( n = 4)

Article Snippet: TFEB shRNA lentiviral particles were from Santa Cruz (sc-38510-V) and control shRNA lentiviral particles were from Origene (TR30037).

Techniques: Cell Culture, Western Blot, Expressing, Immunofluorescence, Translocation Assay, Staining, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

doi: 10.1038/s41419-019-1931-4

Figure Lengend Snippet: a – e SMCs cultured in full-serum medium were treated with trehalose (0–100 mM) for indicated time points. Cell proliferation was analyzed by counting the cell numbers ( a ). Immunoblotting analysis ( b , c ) shows the effects of trehalose (24 h) on the cell cycle protein cyclin D1 and CDK4. d – e Immunofluorescence images show the proliferative cell marker Ki67 (red) in SMCs treated with vehicle or 100 mM trehalose for 24 h. Nuclei were stained with DAPI. f–j SMCs were transfected with scramble or TFEB cDNA plasmids for 24 h and then cultured in full-serum medium for indicated time points. f Cell number counting. g , h Immunoblotting analysis shows the effects of TFEB overexpression on cyclin D1 and CDK4 in SMCs 48 h after transfection. i , j Immunofluorescence images for Ki67 (red) in SMCs transfected SMC SMCs 48 h after transfection. Scale bar = 50 µm * P < 0.05 ( n = 4)

Article Snippet: TFEB shRNA lentiviral particles were from Santa Cruz (sc-38510-V) and control shRNA lentiviral particles were from Origene (TR30037).

Techniques: Cell Culture, Western Blot, Immunofluorescence, Marker, Staining, Transfection, Over Expression

Journal: Cell Death & Disease

Article Title: Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

doi: 10.1038/s41419-019-1931-4

Figure Lengend Snippet: a , b SMCs cultured in full-serum medium were treated with trehalose (0–100 mM) for 24 h. Migration was analyzed by scratch assay. c – e SMCs were treated with vehicle or 100 mM trehalose cultured in full-serum medium, or under starvation condition (Starv, 0.1%FBS) for 24 h. Immunofluorescence images ( c ) show the arrangement of F-actin filaments by phalloidin staining (red). d , e Immunofluorescence analysis of MMP2 expression in SMCs. f–j SMCs were also transfected with scramble or TFEB cDNA plasmids for 24 h and then cultured in full-serum medium for another 24 h. f , g Scratch assay of transfected SMCs. h Immunofluorescence images show the F-actin filaments of transfected SMCs. i , j Immunofluorescence analysis of MMP2 expression in transfected SMCs. Scale bar = 50 µm * P < 0.05 ( n = 4)

Article Snippet: TFEB shRNA lentiviral particles were from Santa Cruz (sc-38510-V) and control shRNA lentiviral particles were from Origene (TR30037).

Techniques: Cell Culture, Migration, Wound Healing Assay, Immunofluorescence, Staining, Expressing, Transfection

Journal: Cell Death & Disease

Article Title: Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

doi: 10.1038/s41419-019-1931-4

Figure Lengend Snippet: SMCs in full-serum medium were transduced with scramble or TFEB shRNA lentiviral particles as described in “Methods”. The cells were treated with vehicle or trehalose (100 mM). a Immunoblot analysis show the effects of TFEB silencing on protein expression of LC3 and p62/SQSTM1 in SMCs with trehalose for 24 h ( n = 6). b Cell number counting after 24-h trehalose ( n = 6). c MMP activity of SMCs after 48-h trehalose by a fluorometric MMP activity assay kit. ( n = 4). d Scratch assay of SMCs with trehalose for 24 h ( n = 6). Scale bar = 100 μm * P < 0.05 vs Scr

Article Snippet: TFEB shRNA lentiviral particles were from Santa Cruz (sc-38510-V) and control shRNA lentiviral particles were from Origene (TR30037).

Techniques: Transduction, shRNA, Western Blot, Expressing, Activity Assay, Wound Healing Assay

Journal: Cell Death & Disease

Article Title: Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

doi: 10.1038/s41419-019-1931-4

Figure Lengend Snippet: The left carotid arteries of mice were partially ligated (ligation) and mice were fed ND or HFD for 2–4 weeks after ligation, the right carotid arteries (unligated) of mice were served as control. a , b Representative IHC analysis of TFEB expression (brown color) in cross sections of carotid arteries. c , d Representative IHC analysis of LC3 expression (brown color) in cross sections of carotid arteries. e , f Representative IHC analysis of LAMP-2A expression (brown color) in cross sections of carotid arteries. Scale bar = 100 μm * P < 0.05 ( n = 6–7)

Article Snippet: TFEB shRNA lentiviral particles were from Santa Cruz (sc-38510-V) and control shRNA lentiviral particles were from Origene (TR30037).

Techniques: Ligation, Control, Expressing

Journal: Cell Death & Disease

Article Title: Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

doi: 10.1038/s41419-019-1931-4

Figure Lengend Snippet: The left carotid arteries of mice were partially ligated (ligation), and after ligation mice were fed HFD and treated with vehicle (PBS) or trehalose (i.p. 2 g/kg, every 2 days) for 4 weeks. a , b Representative IHC analysis of TFEB expression (brown color) in cross sections of PLCAs. c , d Representative IHC analysis of LC3 expression (brown color) in cross sections of PLCAs. e , f Representative IHC analysis of LAMP-2A expression (brown color) in cross sections of PLCAs. Scale bar = 100 μm. * P < 0.05 ( n = 7–8)

Article Snippet: TFEB shRNA lentiviral particles were from Santa Cruz (sc-38510-V) and control shRNA lentiviral particles were from Origene (TR30037).

Techniques: Ligation, Expressing

Journal: Molecular Medicine

Article Title: Modulation of anti-cardiac fibrosis immune responses by changing M2 macrophages into M1 macrophages

doi: 10.1186/s10020-024-00858-z

Figure Lengend Snippet: NaW increases lysosomal pH-mediated Ca 2+ release and thus enhances glucose uptake. A – G IL-4-conditioned bone marrow-derived macrophages (BMDMs) were treated with or without NaW (2 mM) for 12 h. (A) Scl2a1 expression was measured by real-time PCR. B The mean fluorescence intensity (MFI) level of 2NBDG was measured by flow cytometry. C LysoSensor Green-labeled acidic lysosomes were observed under a fluorescence microscope. Scale bar, 20 μm. D The MFI of the LysoSensor probe was measured by flow cytometry. E , F TMEM175 expression was measured by real-time PCR ( E ) and western blotting ( F ). G – I IL-4-conditioned BMDMs were transfected with TMEM175 siRNAs for 12 h, the MFI of the LysoSensor probe was detected by flow cytometry ( G ), and Nos2 expression was determined by real-time PCR ( H ) and western blotting ( I ). J After Tmem175 overexpression in macrophages, the MFI emitted by 2NBDG was measured by flow cytometry. K IL-4-conditioned BMDMs were treated with or without NaW (2 mM) for 12 h, and cytosolic calcium release was measured by flow cytometry. L , M IL-4-conditioned BMDMs were treated with NH 4 Cl for 5 min, the pH value of lysosomes was measured with a microplate reader ( L ), and the cytosolic calcium release rate was measured by flow cytometry ( M ). N The expression of Mcoln1 and Mcoln2 was analyzed by real-time PCR. O The same as presented in ( A ), except cells were pretreated with 10 nM CsA for 1 h. P , Q The same as presented in ( A ), except the expression and location of TFEB were analyzed by real-time PCR ( P ) and immunofluorescence ( Q ) (scale bar, 20 μm). R , S IL-4-conditioned BMDMs were transfected with Tfeb siRNA, Scl2a1 expression was measured by real-time PCR, and the MFI of 2NBDG was measured by flow cytometry. Unless otherwise specified, n = 3 biologically independent experiments. The data are presented as the mean ± SEM. P values were calculated using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Short interfering RNAs (siRNAs) targeting mouse Pygl , Tmem175 , and Tfeb and negative control siRNAs (NC) were purchased from RiboBio (Guangzhou, China). siRNA (50 nM) was transfected into macrophages using LipofectamineTM RNAiMAX Transfection Reagent according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Flow Cytometry, Labeling, Microscopy, Western Blot, Transfection, Over Expression, Immunofluorescence

Journal: PLoS ONE

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

doi: 10.1371/journal.pone.0180892

Figure Lengend Snippet: (A) Real-time PCR results showing increased SCAMP5 level in primary neuronal cultures treated with Rapamycin(100nM), MG132(3μM) or starvation. Values were normalized to control group as relative expression with respect to endogenous control gene actin (mean ±S.E.M.; n = 3; *p<0.05). (B) Dual-luciferase assay showing that TFEB activates from the SCAMP5 promoter. SH-SY5Y cells were transfected with either an empty vector pcDNA3 or Flag-TFEB and a firefly luciferase reporter directed by the human SCAMP5 promoter. Renilla-luciferase reporter was co-transfected as transfection efficiency control. The relative luminescence of Firefly/Renilla was examined 48h later (mean ±S.E.M.; n = 6; *p<0.05). (C) Immunoblots showing increased SCAMP5 level in SH-SY5Y cells transfected with Flag-TFEB (left panel). Right panel shows the quantification of SCAMP5/β-tubulin expression in the immunoblots of three independent experiments (mean ±S.E.M.; n = 3; *p<0.05). (D) The expression of SCAMP5 during autophagy response in TFEB-silenced cells. SH-SY5Y cells were transfected with siRNA against TFEB (TFEB) or scrambled non-specific siRNA as control (NC), and exposed to MG132 for 6 hour before harvest for analysis by real-time PCR. The silencing of the TFEB in SH-SY5Y cells was confirmed in (E) The expression of TFEB and SCAMP5 were normalized to actin and then to the control (mean ±S.E.M.; n = 3; *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT ); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT ); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT ); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT ); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT ).

Techniques: Real-time Polymerase Chain Reaction, Control, Expressing, Luciferase, Transfection, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

doi: 10.1371/journal.pone.0180892

Figure Lengend Snippet: (A) Immunoblots showing quickly increased SCAMP5 in primary neuronal cultures treated with Rapamycin(100nM) or MG132(3μM). Representative results from four independent experiments with similar conclusions were shown. (B) Immunoblots and the quantification (C) showing that SCAMP5 degrades much quicker than β-actin. Primary neuronal cultures were treated with protein synthesis inhibitor Cycloheximide over a timecourse of 24 hours (mean ±S.E.M.; n = 3; **p<0.01). (D) Representative immunoblots showing that proteasome inhibitor MG132 causes a transient increase of exogenous SCAMP5. Primary neuronal cultures were infected with lentiviral vector expressing neuronal specific SCAMP5, and harvested 72 hours later. MG132 was added 2 to 12 hours before harvest. The results represent three independent experiments with similar results. (E) Representative immunoblots showing that autophagy inhibitor BafilomycinA1 causes a transient increase of exogenous SCAMP5 in primary neuronal cultures expressing lenti-viral delivered SCAMP5 as in (D). BafilomycinA1 was added 2 to 12 hours before harvest. The results represent three independent experiments with similar results. (F) SCAMP5 is ubiquitinated as shown by immunoprecipitation. HEK-293T cells were transfected with HA-Ubiquitin, Flag-SCAMP5, or both for 24 hours. One group was treated with MG132 for 6 hours before harvest. The cells were lysed with RIPA buffer and immunoprecipitated using anti-Flag antibody, then immunoblotted with anti-HA antibody.

Article Snippet: The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT ); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT ); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT ); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT ); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT ).

Techniques: Western Blot, Infection, Plasmid Preparation, Expressing, Immunoprecipitation, Transfection, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

doi: 10.1371/journal.pone.0180892

Figure Lengend Snippet: (A) Immunoblots showing increased LC3-II in SCAMP5 over-expressed cells. HEK-293T were transfected with Flag-GFP, Flag-SCAMP1 or Flag-SCAMP5, and harvest 48 hours later. Autophagy inducer Rapamycin (100nM) was added to culture medium for 2 hours before harvest. In SDS-PAGE, the appeared molecular weight of SCAMP5 was about 25kD when the sample was not boiled. Large aggregated bands would form when the sample was boiled, probably due to its property as a membrane protein. (B) HEK-293T cells were transfected with Flag-GFP or Flag-SCAMP5 and harvest 48 hours later. Autophagy blocker Bafilomycin A1 (50nM) was added to culture medium for indicated time before harvest. The protein level of LC3 and p62 was examined by immunoblotting (upper panel). Lower panel shows the quantification of LC3II/actin in three independent experiments, BafilomycinA1 was applied for 3 hours (mean ±S.E.M.; n = 5; *p<0.05, ***p<0.001). (C) Immunoblots showing increased p62 in cells over-expressing SCAMP5. SH-SY5Y cells were transfected with Flag-GFP or Flag-SCAMP5. Lower panel showed the quantification of three independent experiments (mean ±S.E.M.; n = 3; *p<0.05). (D) Immunoblots (upper panel) and quantification (lower panel) of the effect of SCAMP5 knockdown on LC3II and p62 in the absence or the presence of Rapamycin (mean ±S.E.M.; n = 4 independent experiments; *p<0.05, **p<0.01). (E) Immunofluorescent results of mRFP-GFP-LC3 showing that SCAMP5 blocks the fusion of autophagosome and lysosome. HEK-293T cells were transfected with autophagy reporter mRFP-GFP-LC3 and either empty vector or Flag-SCAMP5, one group was treated with FBS free DMEM medium (starvation) for two hours before harvest. Immunofluorescent was performed 24 hours later using mouse anti-Flag primary antibody and donkey anti mouse Alexa Fluor 647 secondary antibody. Confocal microscopy examination confirmed that all the mRFP-GFP-LC3 transfected cells were Flag-SCAMP5 positive(data not show). The acid-sensitive green fluorescence is lost when autophagosomes fuse with lysosomes as shown in the representative image (left panel). The quantification of the number of RFP+GFP+ autophagosome and RFP+GFP- autolysosome was carried out double-blindly in more than 300 cells(mean ±S.E.M.; n = 3; *p≤0.05, **p<0.01) (right panel).

Article Snippet: The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT ); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT ); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT ); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT ); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT ).

Techniques: Western Blot, Transfection, SDS Page, Molecular Weight, Membrane, Expressing, Knockdown, Plasmid Preparation, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

doi: 10.1371/journal.pone.0180892

Figure Lengend Snippet: (A) Immunofluorescent images of rat primary neuronal culture showing the co-localization of endogenous SCAMP5 (red) and α-synuclein (green) using confocal microscopy. (B) SCAMP5 increases the secretion of α-synuclein over time in cultured cells. EGFP-α-synuclein stable overexpressed SH-SY5Y cells were transfected with Flag-GFP or SCAMP5, and harvested 1 to 2 days after transfection. The amount of transfected EGFP-α-synuclein in cell lysate(CL), cell medium(CM), and SDS-insoluble pellet were analyzed by immunoblotting. (C) Quantification of secreted EGFP-α-synuclein in cell medium of SH-SY5Y cells overexpressed with SCAMP5 or control for 24 hours in three independent experiments. (mean ±S.E.M.; n = 3; *p<0.05). (D) MTS assay showing no significant difference in cell viability between control plasmid or SCAMP5 overexpressed cells. SH-SY5Y cells stably expressing EGFP-α-synuclein-WT were transfected with control plasmid or Flag-SCAMP5. 48 hours later, cells were digested, counted, and adjusted to the same concentration, then applied to MTS assay (mean ±S.E.M.; n = 3). (E) Knockdown of SCAMP5 decreases the secreted EGFP-α-synuclein in cell medium. SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with scrambled siRNA or SCAMP5 siRNA. The CL and CM were collected 72 hours later and examined by immunoblotting. Right panel shows the quantification of secreted EGFP-α-synuclein in cell medium of three independent experiments (mean ±S.E.M; n = 3; ***p<0.001). (F) SCAMP5 causes aggregation and secretion of HTT protein. Immunoblots showing HTT protein in cell lysates (cytoplasm and nucleus portion), RIPA-insoluble pellets (extracted with 8M Urea) and cell medium of HEK-293T cells transfected with an empty vector, SCAMP1 or SCAMP5.

Article Snippet: The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT ); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT ); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT ); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT ); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT ).

Techniques: Confocal Microscopy, Cell Culture, Transfection, Western Blot, Control, MTS Assay, Plasmid Preparation, Stable Transfection, Expressing, Concentration Assay, Knockdown

Journal: PLoS ONE

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

doi: 10.1371/journal.pone.0180892

Figure Lengend Snippet: (A) Inhibitors of Golgi-dependent conventional secretion did not block SCAMP5-mediated secretion of α-synuclein. SH-SY5Y cells stably expressing EGFP-α-synuclein-WT or EGFP-α-synuclein-A53T were transfected with Flag-GFP or Flag-SCAMP5. Cell medium and cell lysate were harvested 48 hours later. Six hours before harvested, the exocytosis inhibitor Tetanus toxic (TeNT, 2nM) or BrefeldinA (BFA, 2μM), or DMSO were applied with a medium change. (B) Representative immunofluorescent images (z-projection) showing Golgi fragmentation in SCAMP5 over-expressed SH-SY5Y cells. Cells were transfected with Flag-SCAMP5, and treated with or without Bafilomycin A1 for 6 hours. 48 hours later, cells were fixed and immuno-stained with antibodies against Flag (green) and Golgi marker GM130 (red) or Giantin (red), and examined with confocal microscopy. Arrows indicate fragmented Golgi apparatus. (C) Quantificantion of Golgi fragmentation. The Golgi apparatus were immuno-stained with GM130 antibody and categorized into three types according to the integrity of its structure: normally stacked Golgi (green arrow), slightly collapsed compact Golgi (yellow arrow), and fragmented Golgi (red arrow). The percentages of cells with indicated Golgi morphology in SCAMP5-/+ cells were determined in three independent experiments (total cell number≥300; mean ±S.E.M.; *p<0.05, **p<0.01, ***p<0.001). (D) Immunofluorescent images showing impaired ER-Golgi transport in SCAMP5 over-expressed SH-SY5Y cells. Cells were transfected with Flag-SCAMP5 and immuno-stained with antibodies against Flag (green) and ERGIC53 (red). Representative morphology of accumulated (arrow) ER-Golgi intermediate compartment was as showed. (E) Statistics shows the percentage of cells with impaired ER-Golgi transport in SCAMP5- or SCAMP5+ cells treated with BafilomycinA1 in four independent experiments (total cell number≥300; mean ±S.E.M.; **p<0.01).

Article Snippet: The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT ); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT ); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT ); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT ); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT ).

Techniques: Blocking Assay, Stable Transfection, Expressing, Transfection, Staining, Marker, Confocal Microscopy

Journal: PLoS ONE

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

doi: 10.1371/journal.pone.0180892

Figure Lengend Snippet: (A) Flowchart showing the exosome extraction procedure. (B) Quality control of exosome extraction. Cell lysates(CL), Cell medium(CM), exosome(EXO), Flow through(FT), and RIPA insoluable pellets of EGFP-α-synuclein stable overexpressed SH-SY5Y cells were collected according to the flowchart in 6A. The difference in apparent molecular weight was mainly due to different buffer between samples. Immunoblotting of exosome marker CD63 is conducted using native lysis buffer and native PAGE. Other proteins such as α-synuclein and ER marker Calnexin were examined with SDS-PAGE. (C) Electron microscopy images of exosomes isolated from the cell medium of SH-SY5Y cells stably expressing EGFP-α-synuclein. (D) SCAMP5 is abundant in exosomes, and it increases α-synuclein secretion via exosome. SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with Flag-GFP or Flag-SCAMP5, and harvested 48 hour later. All the samples were processed identically. The loading/total volumes of CL, CM, EXO, and FT were 2/1,200μL, 30/6,000μL, 30/200μL, 30/6,000μL respectively. (E) Quantification of secreted EGFP-α-synuclein in exosome of SH-SY5Y cells overexpressed with SCAMP5 or control in three independent experiments. (mean ±S.E.M.; n = 3; *p<0.05). (F) SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with Flag-GFP or Flag-SCAMP5, and harvested 48 hour later. BrefeldinA was added 6 hours before harvest with a change of medium.

Article Snippet: The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT ); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT ); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT ); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT ); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT ).

Techniques: Extraction, Control, Molecular Weight, Western Blot, Marker, Lysis, Clear Native PAGE, SDS Page, Electron Microscopy, Isolation, Stable Transfection, Expressing, Transfection