Journal: Cell Death and Differentiation

Article Title: SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

doi: 10.1038/s41418-017-0033-z

Figure Lengend Snippet: Effect of Tunicamycin and Hbss treatment on ER–mitochondria contacts. Immunofluorescence against mitochondria (Tom20, red) is shown in the panels on the middle. The green channel is the merge of several planes. Scale bar 20 µm. a Representative confocal pictures of HeLa cells expressing the SPLICS S probe. b Quantification of SPLICS S contacts by 3D rendering of complete z-stacks. Mean ± SEM: Ctrl 58 ± 3, n = 32 cells; Tunicamycin 84 ± 5, n = 33 cells; Hbss 81 ± 5, n = 25 cells. c Representative confocal pictures of HeLa cells expressing the SPLICS L probe. d Quantification of SPLICS L contacts by 3D rendering of complete z-stacks. Mean ± SEM: Ctrl 218 ± 11, n = 27 cells; Tunicamycin 171 ± 9, n = 33 cells; Hbss 204 ± 10, n = 23 cells. Data shown are the result of three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The coverslips were then incubated for 90 min at 37 °C with the specific primary antibody diluted 1:20 in PBS (Tom20: Santa Cruz Biotech., Cat#sc-11415; Parkin: Santa Cruz Biotech., Cat#sc-32282; Drp1: BD Biosciences, Cat#611113; mtHSP60: Abcam, Cat#ab82520; Calreticulin: Thermo Fisher, Cat#PA3-900; Myc: Millipore, Cat#05-724; HA: Cell Signalling, Cat#3724S).

Techniques: Immunofluorescence, Expressing

Journal: Cell Death and Differentiation

Article Title: SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

doi: 10.1038/s41418-017-0033-z

Figure Lengend Snippet: Effects of Drp1 overexpression on ER–mitochondria contacts. Immunofluorescence against mitochondria (Tom20, cyan) and Drp1 (red) is shown in the corresponding panels. The green channel is the merge of several planes. Scale bar 20 µm. a Representative confocal pictures of HeLa cells expressing the SPLICS S probe. b Quantification of SPLICS S contacts by 3D rendering of complete z-stacks. Mean ± SEM: Ctrl 59 ± 3, n = 79 cells; Drp1 WT 70 ± 5, n = 32 cells; Drp1-K38A 89 ± 9, n = 28 cells. c Representative confocal pictures of HeLa cells expressing the SPLICS L probe. d Quantification of SPLICS L contacts by 3D rendering of complete z-stacks. Mean ± SEM: Ctrl 260 ± 14, n = 24 cells; Drp1 WT 198 ± 14, n = 24 cells. Data shown are the result of 3–4 independent experiments. ** p ≤ 0.01.

Article Snippet: The coverslips were then incubated for 90 min at 37 °C with the specific primary antibody diluted 1:20 in PBS (Tom20: Santa Cruz Biotech., Cat#sc-11415; Parkin: Santa Cruz Biotech., Cat#sc-32282; Drp1: BD Biosciences, Cat#611113; mtHSP60: Abcam, Cat#ab82520; Calreticulin: Thermo Fisher, Cat#PA3-900; Myc: Millipore, Cat#05-724; HA: Cell Signalling, Cat#3724S).

Techniques: Over Expression, Immunofluorescence, Expressing

Journal: Cell Death and Differentiation

Article Title: SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

doi: 10.1038/s41418-017-0033-z

Figure Lengend Snippet: Effect of Mfn2 knockdown and mutant PS2 on ER–mitochondria interface. Immunofluorescence against mitochondria (Tom20, red) is shown in the panels on the middle. The green channel is the merge of several planes. Scale bar 20 µm. a Representative confocal pictures of HeLa cells expressing the SPLICS S probe. b Quantification of SPLICS S contacts by 3D rendering of complete z-stacks. Mean ± SEM: SCR shRNA 70 ± 4, n = 76 cells; shRNA Mfn2 #1 98 ± 7, n = 26 cells; shRNA Mfn2 #3 108 ± 7, n = 28 cells; shRNA Mfn2 #4: 95 ± 7, n = 23 cells. c Representative confocal pictures of HeLa cells expressing the SPLICS L probe. d Quantification of SPLICS L contacts by 3D rendering of complete z-stacks. Mean ± SEM: SCR shRNA 238 ± 10, n = 30 cells; shRNA Mfn2 #1 172 ± 9, n = 27 cells; shRNA Mfn2 #3 176 ± 8, n = 29 cells; shRNA Mfn2 #4 190 ± 10, n = 27 cells. e Representative confocal pictures of human fibroblasts from a patient with the N141I mutation in PS2 (bottom panel) and an age-matched control (upper panel) expressing the SPLICS S probe. The green channel is the merge of several planes. Scale bar 20 µm. f Quantification of ER–mitochondria short contacts by 3D rendering of complete z-stacks. Mean ± SEM: CTRL 50 ± 5, n = 20 cells; PS2-N14I: 101 ± 12, n = 21 cells. Data shown are the result of 2–5 independent experiments. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The coverslips were then incubated for 90 min at 37 °C with the specific primary antibody diluted 1:20 in PBS (Tom20: Santa Cruz Biotech., Cat#sc-11415; Parkin: Santa Cruz Biotech., Cat#sc-32282; Drp1: BD Biosciences, Cat#611113; mtHSP60: Abcam, Cat#ab82520; Calreticulin: Thermo Fisher, Cat#PA3-900; Myc: Millipore, Cat#05-724; HA: Cell Signalling, Cat#3724S).

Techniques: Mutagenesis, Immunofluorescence, Expressing, shRNA

Journal: Cell Death and Differentiation

Article Title: SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

doi: 10.1038/s41418-017-0033-z

Figure Lengend Snippet: Effects of Parkin on ER–mitochondria contacts. Immunofluorescence against mitochondria (Tom20, cyan) is shown in the panels on the middle. The green channel is the merge of several planes. Scale bar 20 µm. a Representative confocal pictures of HeLa cells expressing the SPLICS S probe along with Parkin-2A-mCherry-CAAX (bottom panels). b Quantification of SPLICS S contacts by 3D rendering of complete z-stacks. Mean ± SEM: Ctrl 61 ± 3, n = 57 cells; Parkin 94 ± 6, n = 33 cells; Parkin CCCP 65 ± 7, n = 26 cells. c Representative confocal pictures of HeLa cells expressing the SPLICS L probe along with Parkin-2A-mCherry-CAAX (bottom panels). d Quantification of the SPLICS L contacts by 3D rendering of complete z-stacks. Mean ± SEM: CTRL 227 ± 9, n = 44 cells; Parkin 193 ± 11, n = 32 cells; Parkin CCCP 159 ± 8, n = 22 cells. Data shown are the result of 3–8 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: The coverslips were then incubated for 90 min at 37 °C with the specific primary antibody diluted 1:20 in PBS (Tom20: Santa Cruz Biotech., Cat#sc-11415; Parkin: Santa Cruz Biotech., Cat#sc-32282; Drp1: BD Biosciences, Cat#611113; mtHSP60: Abcam, Cat#ab82520; Calreticulin: Thermo Fisher, Cat#PA3-900; Myc: Millipore, Cat#05-724; HA: Cell Signalling, Cat#3724S).

Techniques: Immunofluorescence, Expressing