Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 enhances lysosomal calcium levels. (A-B) Vector/HT1080 and TMBIM6/HT1080 (A) or Tmbim6 +/+ and tmbim6 −/- MEF cells (B) were treated with the indicated agent, and Fura-2 Ca 2+ imaging was performed as described in Materials and Methods. (C-D) ER Ca 2+ stores were emptied with 1–5 μM thapsigargin or and 1 μM ionomycin before inducing Ca 2+ release from acidic stores by GPN in vector/HT1080 and TMBIM6/HT1080 cells (C) or Tmbim6 +/+ and tmbim6 −/- MEF cells (D). (E-F) Fluorescence images of intralumenal Ca 2+ in vector/HT1080 and TMBIM6/HT1080 cells. Representative images were showing the cells loaded with low-affinity Rhod-dextran (LA-RhodDx, E) and OG-BAPTA-dextran (F). Scale bar: 15 µm. (G) Time-lapse images of GPN-treated GCaMP3-ML1-expressing vector/HT1080 and TMBIM6/HT1080 cells during the indicated time periods. The GPN responses were quantified (bottom). Representative fluorescence image of GCaMP3-ML1-expressing (green) and LysoTracker-loaded (red) vector/HT1080 and TMBIM6/HT1080 cells (up, right). The data are represented as mean ± SEM from n = 3 independent experiments

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: Plasmid Preparation, Imaging, Fluorescence, Expressing

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 regulates the lysosomal calcium level. (A) Fluorescence images of intralumenal Ca 2+ in HT1080 cells loaded with OG-BAPTA-dextran using siRNA of TMBIM6 ( TMBIM6 RNAi) or ITPR ( ITPR RNAi). Scale bar: 15 µm. The right graph represents the fold of fluorescence intensity under indicated siRNA transfection compared with cells transfected with scrambled siRNA oligonucleotides. (B) qPCR analysis of TMBIM6 and ITPR (bottom) was performed to confirm the efficacy of siRNA-mediated silencing. (C) Fura-2 AM was loaded in HT1080 cells with TMBIM6 and ITPR RNAi, treated with GPN (200 μM), and then the difference between the peak value upon the treatment and the resting value before treatment was analyzed. (D) In HT1080 cells, stably expressing GCaMP3-ML1 with TMBIM6 or ITPR RNAi, the ML1 channel agonist ML-SA1 (25 μM) was added in a Ca 2+ -free external solution for increasing GCaMP3 fluorescence (F470). Note that we typically set F0 based on the value that is closest to the baseline (up to 10 min). (E) Proximity ligation assay (PLA) between ZFYVE27/protrudin (ER membrane) and RAB7A/RAB7 (endolysosome) in cells overexpressing TMBIM6 and vector cells. Scale bar: 15 µm. The data are represented as the mean ± SEM from n = 3 independent experiments. Asterisks indicate significant differences from the scramble. Hash indicates significant differences between double depleted and other cells. (A, C, D) One-way ANOVA

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: Fluorescence, Transfection, Stable Transfection, Expressing, Proximity Ligation Assay, Plasmid Preparation

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 functions as a calcium-permeating channel. (A) Intracellular Ca 2+ was recorded in Fura-2AM-loaded empty, TMBIM6 WT, or TMBIM6 D213A HT1080 cells. (B) The intensities of fluorescence measured in TMBIM6-GCaMP3 WT- and TMBIM6-GCaPM3 D213A -overexpressing HT1080 cells (F470). (C) Thapsigargin (5 μM) in a Ca 2+ -free external solution was applied to TMBIM6-GCaMP3 WT- and TMBIM6-GCaPM3 D213A -overexpressing HT1080 cells. (D) Fluorescence images of intralumenal Ca 2+ in empty vector-, TMBIM6 WT-, and TMBIM6 D213A HT1080 cells loaded with OG-BAPTA-dextran. Scale bar: 15 µm. The data are represented as mean ± SEM from n = 3 independent experiments (right). (E) Intracellular Ca 2+ was recorded in Fura-2AM-loaded empty vector, TMBIM6 WT, or TMBIM6 D213A HT1080 cells induced by GPN. The data are represented as mean ± SEM from n = 3 independent experiments (right)

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: Fluorescence, Plasmid Preparation

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 increases lysosomal calcium release through MCOLN1 under stress conditions. (A) Fura-2AM-loaded GCaMP3-ML1 cells transfected with TMBIM6 RNAi were treated with ML-SA1 (25 μM) in a Ca 2+ -free external solution under starvation or torin or PP242 treatment for 1 h. Ca 2+ concentrations are based on the calibration of the Fura-2 signal. The bar graph shows the difference between the peak value upon the addition of agonist and the resting value before the addition of agonist. The data are the means ± SEM from n = 3 independent experiments (bottom). (B) qPCR analysis of TMBIM6 was performed to confirm the efficacy of siRNA-mediated silencing

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: Transfection

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 enhances TFEB nuclear localization independent of MTORC1 activity. (A) Proximity ligation assay (PLA) between TFEB and PPP3CA (red dots) in TMBIM6- or TMBIM6 D213A expressing HT1080 cells and vector cells under starvation or torin or PP242-treatment. Scale bar: 15 µm. The data are represented as mean ± SEM from n = 3 independent experiments (right). (B) Fluorescence images of endogenous TFEB after 3 h of starvation or torin or PP242-treatment TFEB nuclear translocation. Scale bar: 15 µm. The data are represented as the mean ± SEM from n = 3 independent experiments. (C) Fluorescence images of endogenous TFEB in siRNA of TMBIM6 and MCOLN1 -pretreated cells under starvation or torin or PP242-treatment after 3 h. Scale bar: 15 µm. (D) qPCR analysis of TMBIM6 and MCOLN1 was performed to confirm the efficacy of siRNA-mediated silencing. Asterisks indicate significant differences from vector or scramble siRNA treatments. The hash indicates significant differences between TMBIM6 and TMBIM6 D213A

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: Activity Assay, Proximity Ligation Assay, Expressing, Plasmid Preparation, Fluorescence, Translocation Assay

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 enhances autophagy flux. (A) Lysosomal staining was performed with 100 nM LysoTracker for 30 min. Vector, TMBIM6, and TMBIM6 D213A /HT1080 cells were starved or treated with torin1 or PP242 for 3 h. The data are represented as the mean ± SEM from n = 3 independent experiments (bottom). Scale bar, 25 µm. (B) Autophagic flux was determined using cyto-ID under microplate reader. (C) Immunoblotting of cell lysates against LC3B and SQSTM1 was performed and quantified (bottom). N, vector; B, TMBIM6; M, TMBIM6 D213A . (D) RFP-GFP-LC3 puncta formation was analyzed in vector, TMBIM6, and TMBIM6 D213A /HT1080 cells under starvation or torin or PP242-treatment for 3 h. The yellow puncta (autophagosome) and red puncta (autolysosome) formation were quantified (bottom). Scale bar: 15 μm. The data are represented as the mean ± SEM from n = 3 independent experiments. Asterisks indicate significant differences from the vector treatment. Hash indicates significant differences between TMBIM6 and TMBIM6 D213A

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: Staining, Plasmid Preparation, Western Blot

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 increases autophagy flux under starvation conditions in vivo . (A-B) GFP-LC3 puncta formation was analyzed in the liver or kidney (A) of nutrient-starved GFP-LC3/ Tmbim6 +/+ and GFP-LC3/ tmbim6 −/- mice after 24 h (n = 5). GFP-LC3 puncta formation was quantified (B). Scale bar: 100 μm. The data are represented as the mean ± SEM from n = 5 per group. (C-D) Immunohistochemistry analysis of SQSTM1 was performed in the kidney and liver from control and 24-h starved Tmbim6 +/+ and tmbim6 −/- mice (C). The SQSTM1 expression levels were quantified (D). Scale bar: 100 μm. The data are represented as the mean ± SEM from n = 5 per group. (E) HT1080 cells were transfected with or without TMBIM6 or ITPR siRNA together with EGFP-HTTQ74 for 24 h. The percentage of cells with EGFP-positive aggregates is shown. Scale bar: 25 µm. (F) EGFP-HTTQ74 clearance was analyzed in transfected HT1080 cells vector, TMBIM6, and TMBIM6 D213A cells. Scale bar: 25 µm. The data are represented as the mean ± SEM from n = 3 independent experiments

Article Snippet: The fresh puromycin-containing medium was replaced every 3 to 4 d. Established HT1080 cell lines were as follows: pLenti CMV/TO puro DEST (670–1; vector HT1080 cells), pLenti CMV/TO puro DEST (670-1)-TMBIM6 (TMBIM6 HT1080 cells), and pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3 HT1080 cells). pLenti CMV/TO puro DEST (670–1) were obtained from Addgene (#17293).

Techniques: In Vivo, Immunohistochemistry, Expressing, Transfection, Plasmid Preparation