tfeb-gfp expression vector  (Orbigen Inc)

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: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: TFEB translocates to the nucleus during mitophagy in a Parkin- and PINK1-dependent manner. (A) YFP-Parkin HeLa cells were treated with O/A for up to 10 h, fractionated, and immunoblotted. (B) Quantification of data in A. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (C) mCherry-Parkin HeLa cells were left untreated (Control), starved (2 h), or treated with torin 1 (2 h), O/A (6 h), or valinomycin (Val; 6 h). CIP treatment of cell lysates was performed before immunoblotting. (D) WT and mCherry-Parkin HeLa cells were treated with DMSO or O/A (6 h), lysed, and immunoblotted. A CIP-treated control was included as a reference for total TFEB dephosphorylation. (E) WT and mCherry-Parkin HeLa cells were treated with DMSO, torin 1, or O/A for 18 h and analyzed by quantitative PCR for TFEB target gene expression. Data are means ± SD ( n = 3). (F) WT and PINK1 KO HeLa cells stably expressing TFEB-GFP with or without mCherry-Parkin were treated as in C. Fixed cells were stained with DAPI and analyzed by immunofluorescence. Bars, 10 µm. See Fig. S1 F for quantification. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Expressing, Western Blot, De-Phosphorylation Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Staining, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Analysis of Parkin-dependent effects on mTORC1 activity and TFEB association with 14-3-3 proteins. (A and C) WT and YFP-Parkin HeLa cells were treated with DMSO, O/A, or torin 1 as indicated, lysed, and immunoblotted. (B and D) Quantification of data in A and C, respectively. Protein levels were normalized to actin and the ratio of phosphorylated to total protein in treated samples is expressed relative to DMSO controls. Data are means ± SD ( n = 3); no differences observed were statistically significant. (E) HeLa cells stably expressing TFEB-GFP were transfected with control or untagged Parkin DNA and treated the next day with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Cells were lysed and TFEB-GFP was immunoprecipitated with anti-GFP beads. Cell lysates (Input) and immunoprecipitated proteins were immunoblotted. Images are representative of n = 2 experiments.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Parkin acts upstream of Rag GTPases to regulate TFEB subcellular localization. (A and B) HeLa cells stably expressing TFEB-GFP with (B) or without (A) mCherry-Parkin were transfected with empty vector or active or inactive RagB/D heterodimer DNA (detected with αHA antibody). The indicated cells were starved (2 h) or treated with DMSO or O/A (6 h) before fixation and immunofluorescence analysis. Images are representative of n = 2 experiments. 75–100% of cells observed (mean of 175 cells per condition) exhibited the given phenotype. Asterisks indicate αHA-negative cells. Bars, 10 µm.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Atg5 and Atg9A are required for Parkin-mediated TFEB translocation. (A) WT and Atg5 KO cells stably expressing mCherry-Parkin were treated with DMSO or O/A (6 h), fixed, immunostained for TFEB, and analyzed by immunofluorescence. Bars, 10 µm. (B) Quantification of endogenous TFEB nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) Untreated WT and Atg5 KO cells stably expressing mCherry-Parkin and GFP-Atg5 as indicated were lysed and immunoblotted. (D) Cells from C were treated with DMSO or O/A (6 h), lysed, fractionated, and immunoblotted. (E) Quantification of data in D. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB was expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (F) WT and Atg5 KO HeLa cells stably expressing mCherry-Parkin as indicated were treated with DMSO (6 h), torin 1 (2 h), CIP (1 h), and O/A (6 h) as indicated, lysed, and immunoblotted. Images are representative of n = 3 experiments. (G) WT and Atg5 KO cells expressing mCherry-Parkin as indicated were starved (2 h) or left untreated, lysed, fractionated, and immunoblotted. Images are representative of n = 3 experiments. (H) WT and Atg9A KO HeLa cells expressing mCherry-Parkin as indicated were starved (2 h), or treated with DMSO (Ctrl) or O/A for 6 h. Cell lysates were processed as in D. (I) Quantification of endogenous TFEB nuclear localization in H, performed as in E. Data are means ± SD ( n = 3). C, cytosol; N, nuclear. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Translocation Assay, Stable Transfection, Expressing, Immunofluorescence, Fluorescence

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Parkin- and Atg5-dependent regulation of TFEB homologue subcellular localization. (A) WT HeLa cells stably expressing mCherry-Parkin, TFE3-GFP, MITF1-GFP, and TFEC-YFP as indicated were treated with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Fixed cells were analyzed by immunofluorescence. (B) Quantification of ectopic TFE3, MITF1, and TFEC nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) WT and Atg5 KO cells stably expressing mCherry-Parkin treated with DMSO or O/A (6 h) were fixed, immunostained for TFE3 or MITF, and analyzed by immunofluorescence. (D) Quantification of endogenous TFE3 and MITF nuclear localization in C. Analysis was performed as in B (40–60 cells/field, 4 fields/condition, n = 3 experiments). Data are means ± SD. For all graphs: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Fluorescence