Journal: Autophagy

Article Title: Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine

doi: 10.1080/15548627.2022.2063003

Figure Lengend Snippet: Overexpression of SIGMAR1/Sigma-1 receptor increased nuclear TFEB level in (G 4 C 2 ) 31 -RNA repeat-transfected NSC34 cells. ( A ) TFEB translocation in nucleus under SIGMAR1-expressing in (G 4 C 2 ) 31 -RNA repeat NSC-34 cells. Confocal images demonstrated GFP-TFEB colocalizes with DAPI in NSC34 cells. ( B ) The quantification data from (A) showed an increased nuclear GFP-TFEB intensity. Intensity analyses were performed by using NIH ImageJ. (version 1.51b). Note: Data shown are percentages of “Average nuclear fluorescence intensity/Average whole cell fluorescence intensity” for each group. HA groups N = 42; HA-SIGMAR1 groups N = 34; two-tailed unpaired Students t test, *** * p < 0.0001. ( C ) Overexpression of HA-SIGMAR1 increased the nuclear TFEB expression caused by the EGFP-(G4C2) 31 . Analyses of western blot showed that the overexpression of HA-SIGMAR1 increased the protein level of nuclear TFEB and concomitantly decreased the cytoplasmic TFEB caused by GFP-(G4C2) 31 . Quantitative data are means ± SEM; N = 4; two-tailed unpaired Students t test, p = 0.0814 (cytosolic TFEB), p = 0.0323 (nuclear TFEB), * p < 0.05.

Article Snippet: NSC34 cells were transfected with GFP-TFEB vector (Origene, MR223018L4) in 10% FBS DMEM medium overnight.

Techniques: Over Expression, Transfection, Translocation Assay, Expressing, Fluorescence, Two Tailed Test, Western Blot

Journal: Autophagy

Article Title: Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine

doi: 10.1080/15548627.2022.2063003

Figure Lengend Snippet: Overexpression of POM121 rescues the TFEB translocation into nucleus in (G 4 C 2 ) 31 -RNA repeat-treated NSC34 cells. ( A ) Increased level of nuclear GFP-TFEB in POM121-overexpressing, (G 4 C 2 ) 31 -RNA repeat-treated NSC34 cells. Confocal images demonstrated the GFP-TFEB colocalization with DAPI in NSC34 cells. ( B ) The quantification of data from (A) showed a significant increase in the intensity of nuclear GFP-TFEB. The intensity analysis was performed by using NIH ImageJ. (version 1.51b). Note: Data shown are percentages of “Average nuclear fluorescence intensity/Average whole cell fluorescence intensity” for each group; MYC/DDK group, N = 35; POM121-MYC/DDK group, N = 24; two-tailed unpaired Students t test, **** p < 0.0001. ( C ) Analyses of western blot shows that the overexpression of POM121 increased the protein level of nuclear TFEB and concomitantly decreased the cytoplasmic TFEB caused by GFP-(G4C2) 31 . Quantitative data are means ± SEM; N = 3; two-tailed unpaired Students t test, p = 0.0060 (cytoplasmic TFEB), p = 0.0407 (nuclear TFEB), * p < 0.05, ** p < 0.01.

Article Snippet: NSC34 cells were transfected with GFP-TFEB vector (Origene, MR223018L4) in 10% FBS DMEM medium overnight.

Techniques: Over Expression, Translocation Assay, Fluorescence, Two Tailed Test, Western Blot

Journal: Autophagy

Article Title: Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine

doi: 10.1080/15548627.2022.2063003

Figure Lengend Snippet: Pridopidine treatment rescued nuclear TFEB level in (G 4 C 2 ) 31 -RNA repeats-treated NSC34 cells. ( A ) Nuclear GFP-TFEB level was increased by pridopidine in (G 4 C 2 ) 31 -RNA repeated-NSC34 cells. Confocal images demonstrate the GFP-TFEB colocalization with DAPI in NSC34 cells. ( B ) The quantification data from (A) showed an increased intensity of GFP-TFEB in the nucleus. Intensity analysis was performed by using NIH ImageJ. (version 1.51b). Note: Data shown are percentages of “Average nuclear fluorescence intensity/Average whole cell fluorescence intensity” for each group. Control groups, N = 28; pridopidine treatment groups, N = 21; two-tailed unpaired Students t test, p = 0.0012, **** p < 0.0001. ( C ) Analyses of shows that pridopidine treatment increased the nuclear TFEB protein expression and decreased the cytoplasmic TFEB caused by the GFP-(G4C2) 31 . Quantitative data are means ± SEM; N = 3; two-tailed unpaired Students t test, p = 0.0050 (cytoplasmic TFEB), p = 0.0011 (nuclear TFEB), ** p < 0.01.

Article Snippet: NSC34 cells were transfected with GFP-TFEB vector (Origene, MR223018L4) in 10% FBS DMEM medium overnight.

Techniques: Fluorescence, Two Tailed Test, Expressing

Journal: Autophagy

Article Title: Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine

doi: 10.1080/15548627.2022.2063003

Figure Lengend Snippet: List of antibodies, cDNA plasmid vectors, and oligonucleotide sequences. WB: western blot; IF: immunofluorescence; IP: immunoprecipitation.

Article Snippet: NSC34 cells were transfected with GFP-TFEB vector (Origene, MR223018L4) in 10% FBS DMEM medium overnight.

Techniques: Plasmid Preparation, Western Blot, Immunofluorescence, Immunoprecipitation, Transduction, shRNA, CRISPR, Real-time Polymerase Chain Reaction

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