Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet: ( A–I ) Cultured astrocytes were co-transfected with 20 μM non-targeting (CTRL) siRNA or Herp siRNA together with G-CEPIA1er ( A–C ), R-GECO1 ( D–F ) or mito-R-GECO1 ( G–I ). At 48 hr post transfection, cultured astrocytes were treated with 100 µM ATP and Ca 2+ imaging analysis was performed. Images were acquired every 3 seconds. ( A, D, G ) Representative time-lapse images of each Ca 2+ indicator. ( B, E, H ) ΔF/F 0 values over time following ATP application. ( C, F, I ) Area above or area under the curve values, calculated from panels B, E, and H. ( A–C ) CTRL siRNA, n=19; Herp siRNA, n=22. ( D–F ) CTRL siRNA, n=20; Herp siRNA, n=25. ( G–I ) CTRL siRNA, n=16; Herp siRNA, n=16. ( J–M ) Cultured astrocytes were transfected with the indicated siRNA (20 nM) and processed for Western blot analysis 48 hr post transfection. Vinculin and GAPDH served as loading control for ITPRs and HERP, respectively. ( J ) Representative western blot images from twelve independent experiments are shown. NS, non-specific band ( K ) Densitometric quantification of western blot data showing relative levels of ITPR1 in Herp siRNA-transfected astrocytes compared to CTRL siRNA transfected astrocytes. ( L ) Representative Western blot images from five independent experiments. ( M ) Densitometric quantification of western blot data showing relative levels of ITPR2 in Herp siRNA-transfected astrocytes compared to Control astrocytes. Values are mean ± SEM (*p<0.05, * * p<0.005, ** * p<0.0005, *** * p<0.00005; t -test). ( N–P ) Cultured astrocytes were treated with 10 μM Xestospongin C (XesC), an IP3R inhibitor, for 30 min before live imaging. Cells were then treated with 100 μM ATP, and images were captured every 3 s. ( N ) Representative time-lapse images of ER Ca 2+ indicator. ( O ) ΔF/F 0 values over time following ATP application. ( P ) Area above the curve values were calculated from panel O. CTRL siRNA + Mock, n=9; Herp siRNA + Mock, n=9; CTRL siRNA + XesC, n=8; Herp siRNA + XesC, n=14. Values are means ± SEM (*p<0.05, * * p<0.005, ** * p<0.0005, *** * p<0.00005; one-way ANOVA). Figure 3—source data 1. PDF file containing original western blot for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in . Figure 3—source data 3. PDF file containing original western blot for , indicating the relevant bands and treatments. Figure 3—source data 4. Original files for western blot analysis displayed in .

Article Snippet: Membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: anti-BMAL1 (Abcam (UK), ab93806), 1:2000; anti-HERP (Abcam, ab150424), 1:1000; anti-ITPR1 (Alomone Labs, Israel, ACC-019), 1:1000; anti-ITPR2 (Alomone Labs, ACC-116), 1:1000; anti-CX43 (Sigma, C6219), 1:5000; anti-pCX43 (Ser368; CST, USA, 3511), 1:1000; anti-GAPDH (Novus, USA, NB100-56875), 1:5000; anti-Vinculin (Sigma-Aldrich, V4505), 1:5000; and anti-total ERK (CST, 9102), 1:5000.

Techniques: Cell Culture, Transfection, Imaging, Western Blot, Control

Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet: ( A ) Schematic diagram of the experimental scheme from transfection to live-cell Ca 2+ imaging at different times. ( B–M ) Cultured astrocytes were transfected with G-CEPIA1er ( B–D, K–M ), R-GECO1 ( E–G ), or mito-R-GECO1 ( H–J ) compartment-specific Ca 2+ indicators (denoted at left) and then their circadian rhythm was synchronized by SS. ( K–M ) The indicated siRNA was co-transfected with the ER Ca 2+ indicator. After transfection, cells were allowed 48 hr for the siRNA to take effect and stabilize before synchronization by serum shock. At the indicated times, astrocytes were treated with 100 µM ATP and Ca 2+ imaging was performed. ( B, E, H, K ) Representative time-lapse images of each Ca 2+ indicator. ( C, F, I, L ) ΔF/F 0 values over time following ATP application. ( D, G, J, M ) Area above or area under the curve values, calculated from panels C, F, I, and L. ( B–D ) 30 hr post sync, n=24; 42 hr post sync, n=19. ( E–G ) 30 hr post sync, n=33; 42 hr post sync, n=38. ( H–J ) 30 hr post sync, n=50; 42 hr post sync, n=54. ( K–M ) CTRL siRNA, 30 hr post sync, n=5; CTRL siRNA, 42 hr post sync, n=4; Herp siRNA, 30 hr post sync, n=11; Herp siRNA, 42 hr post sync, n=5. Values in graphs are mean ± SEM (*p<0.05, *** * p<0.00005); ( D, G, J ) t -test, ( M ) one-way ANOVA. ( N–O ) Cells were harvested at the indicated times and processed for western blot analysis. Vinculin and GAPDH served as loading controls for ITPR and BMAL1, respectively. ( N ) Representative western blot images from six independent experiments. ( O ) Densitometric quantification of western blot data showing relative levels of ITPR1 and ITPR2 at different times. Values in graphs are mean ± SEMs (*p<0.05, *** * p<0.00005; t -test). Panel A was created with BioRender.com . Figure 4—source data 1. PDF file containing original western blot for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: anti-BMAL1 (Abcam (UK), ab93806), 1:2000; anti-HERP (Abcam, ab150424), 1:1000; anti-ITPR1 (Alomone Labs, Israel, ACC-019), 1:1000; anti-ITPR2 (Alomone Labs, ACC-116), 1:1000; anti-CX43 (Sigma, C6219), 1:5000; anti-pCX43 (Ser368; CST, USA, 3511), 1:1000; anti-GAPDH (Novus, USA, NB100-56875), 1:5000; anti-Vinculin (Sigma-Aldrich, V4505), 1:5000; and anti-total ERK (CST, 9102), 1:5000.

Techniques: Transfection, Imaging, Cell Culture, Western Blot

Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet:

Article Snippet: Membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: anti-BMAL1 (Abcam (UK), ab93806), 1:2000; anti-HERP (Abcam, ab150424), 1:1000; anti-ITPR1 (Alomone Labs, Israel, ACC-019), 1:1000; anti-ITPR2 (Alomone Labs, ACC-116), 1:1000; anti-CX43 (Sigma, C6219), 1:5000; anti-pCX43 (Ser368; CST, USA, 3511), 1:1000; anti-GAPDH (Novus, USA, NB100-56875), 1:5000; anti-Vinculin (Sigma-Aldrich, V4505), 1:5000; and anti-total ERK (CST, 9102), 1:5000.

Techniques: Transfection, Construct, Plasmid Preparation, Sequencing, Negative Control, Software, Microscopy

Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet: ( A–I ) Cultured astrocytes were co-transfected with 20 μM non-targeting (CTRL) siRNA or Herp siRNA together with G-CEPIA1er ( A–C ), R-GECO1 ( D–F ) or mito-R-GECO1 ( G–I ). At 48 hr post transfection, cultured astrocytes were treated with 100 µM ATP and Ca 2+ imaging analysis was performed. Images were acquired every 3 seconds. ( A, D, G ) Representative time-lapse images of each Ca 2+ indicator. ( B, E, H ) ΔF/F 0 values over time following ATP application. ( C, F, I ) Area above or area under the curve values, calculated from panels B, E, and H. ( A–C ) CTRL siRNA, n=19; Herp siRNA, n=22. ( D–F ) CTRL siRNA, n=20; Herp siRNA, n=25. ( G–I ) CTRL siRNA, n=16; Herp siRNA, n=16. ( J–M ) Cultured astrocytes were transfected with the indicated siRNA (20 nM) and processed for Western blot analysis 48 hr post transfection. Vinculin and GAPDH served as loading control for ITPRs and HERP, respectively. ( J ) Representative western blot images from twelve independent experiments are shown. NS, non-specific band ( K ) Densitometric quantification of western blot data showing relative levels of ITPR1 in Herp siRNA-transfected astrocytes compared to CTRL siRNA transfected astrocytes. ( L ) Representative Western blot images from five independent experiments. ( M ) Densitometric quantification of western blot data showing relative levels of ITPR2 in Herp siRNA-transfected astrocytes compared to Control astrocytes. Values are mean ± SEM (*p<0.05, * * p<0.005, ** * p<0.0005, *** * p<0.00005; t -test). ( N–P ) Cultured astrocytes were treated with 10 μM Xestospongin C (XesC), an IP3R inhibitor, for 30 min before live imaging. Cells were then treated with 100 μM ATP, and images were captured every 3 s. ( N ) Representative time-lapse images of ER Ca 2+ indicator. ( O ) ΔF/F 0 values over time following ATP application. ( P ) Area above the curve values were calculated from panel O. CTRL siRNA + Mock, n=9; Herp siRNA + Mock, n=9; CTRL siRNA + XesC, n=8; Herp siRNA + XesC, n=14. Values are means ± SEM (*p<0.05, * * p<0.005, ** * p<0.0005, *** * p<0.00005; one-way ANOVA). Figure 3—source data 1. PDF file containing original western blot for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in . Figure 3—source data 3. PDF file containing original western blot for , indicating the relevant bands and treatments. Figure 3—source data 4. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Rabbit polyclonal anti-ITPR2 , Alomone Labs , Cat# ACC-116; Lot: ACC116AN015; RRID: AB_2340910 , 1:1000.

Techniques: Cell Culture, Transfection, Imaging, Western Blot, Control

Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet: ( A ) Schematic diagram of the experimental scheme from transfection to live-cell Ca 2+ imaging at different times. ( B–M ) Cultured astrocytes were transfected with G-CEPIA1er ( B–D, K–M ), R-GECO1 ( E–G ), or mito-R-GECO1 ( H–J ) compartment-specific Ca 2+ indicators (denoted at left) and then their circadian rhythm was synchronized by SS. ( K–M ) The indicated siRNA was co-transfected with the ER Ca 2+ indicator. After transfection, cells were allowed 48 hr for the siRNA to take effect and stabilize before synchronization by serum shock. At the indicated times, astrocytes were treated with 100 µM ATP and Ca 2+ imaging was performed. ( B, E, H, K ) Representative time-lapse images of each Ca 2+ indicator. ( C, F, I, L ) ΔF/F 0 values over time following ATP application. ( D, G, J, M ) Area above or area under the curve values, calculated from panels C, F, I, and L. ( B–D ) 30 hr post sync, n=24; 42 hr post sync, n=19. ( E–G ) 30 hr post sync, n=33; 42 hr post sync, n=38. ( H–J ) 30 hr post sync, n=50; 42 hr post sync, n=54. ( K–M ) CTRL siRNA, 30 hr post sync, n=5; CTRL siRNA, 42 hr post sync, n=4; Herp siRNA, 30 hr post sync, n=11; Herp siRNA, 42 hr post sync, n=5. Values in graphs are mean ± SEM (*p<0.05, *** * p<0.00005); ( D, G, J ) t -test, ( M ) one-way ANOVA. ( N–O ) Cells were harvested at the indicated times and processed for western blot analysis. Vinculin and GAPDH served as loading controls for ITPR and BMAL1, respectively. ( N ) Representative western blot images from six independent experiments. ( O ) Densitometric quantification of western blot data showing relative levels of ITPR1 and ITPR2 at different times. Values in graphs are mean ± SEMs (*p<0.05, *** * p<0.00005; t -test). Panel A was created with BioRender.com . Figure 4—source data 1. PDF file containing original western blot for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Rabbit polyclonal anti-ITPR2 , Alomone Labs , Cat# ACC-116; Lot: ACC116AN015; RRID: AB_2340910 , 1:1000.

Techniques: Transfection, Imaging, Cell Culture, Western Blot

Journal: eLife

Article Title: Circadian regulation of endoplasmic reticulum calcium response in cultured mouse astrocytes

doi: 10.7554/eLife.96357

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-ITPR2 , Alomone Labs , Cat# ACC-116; Lot: ACC116AN015; RRID: AB_2340910 , 1:1000.

Techniques: Transfection, Construct, Plasmid Preparation, Sequencing, Negative Control, Software, Microscopy

Journal: eLife

Article Title: Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation

doi: 10.7554/eLife.80273

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-IP3 receptor 2 (rabbit) (Itpr2) , Millipore , AB3000 , 1:40.

Techniques: Electron Microscopy, In Situ, Protease Inhibitor, Bradford Assay, Membrane, Western Blot, Plasmid Preparation