Journal: The Journal of Biological Chemistry

Article Title: mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

doi: 10.1074/jbc.RA118.002800

Figure Lengend Snippet: Inhibition of PI3K/Akt/mTORC1 signals promotes GSK3β nuclear localization. A, representative images obtained by wide-field epifluorescence microscopy of control RPE cells (no inhibitor treatment) stained to detect endogenous GSK3β or c-Myc, with DAPI stain to identify the nucleus. Scale bar, 20 μm. B–D, RPE cells were treated with either 10 μm LY294002, 5 μm Akti-1/2, or 1 μm rapamycin for 1 h. Following this treatment, cells were then fixed and stained to detect endogenous GSK3β (B) or GSK3α (D). Shown for each (left panels) are micrographs obtained by wide-field epifluorescence microscopy representative of three independent experiments. Scale bar, 20 μm. Also shown for each condition as GSK3β (or α) overlay are sample cellular and nuclear outlines, and a box corresponding to a magnified image of a single cell. Also shown (right panel) is the mean GSK3β or GSK3α nuclear localization index ± S.E. (error bars) (n = 3, >30 cells/condition/experiment); *, p < 0.05 relative to control conditions (absence of LY294002, Akti1/2, and rapamycin). Shown in C are Western blots of cytosolic and nuclear fractions and mean nuclear GSK3β values ± S.E. (n = 3). *, p < 0.05.

Article Snippet: Fluorescence microscopy Wide-field epifluorescence was performed on an Olympus IX83 inverted microscope with a ×100 objective, coupled to a Hamamatsu ORCA-Flash4.0 digital camera (Olympus Canada, Richmond Hill, Canada).

Techniques: Inhibition, Epifluorescence Microscopy, Control, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

doi: 10.1074/jbc.RA118.002800

Figure Lengend Snippet: Rapamycin-induced GSK3β nuclear localization is Ran-dependent. RPE cells were transfected with plasmids encoding HA-tagged WT, T24N, or G19V Ran and then treated with 1 μm rapamycin for 1 h, followed by detection of endogenous GSK3β and exogenous HA-tagged Ran proteins. Shown (top) are micrographs obtained by wide-field epifluorescence microscopy representative of three independent experiments. Scale bar, 20 μm. Also shown for each condition as GSK3β overlay are sample cellular and nuclear outlines and a box corresponding to a magnified image of a single cell. Also shown (bottom) is the mean GSK3β nuclear localization index ± S.E. (error bars) (n = 3, >30 cells/condition/experiment); *, p < 0.05 relative to control conditions (no rapamycin treatment).

Article Snippet: Fluorescence microscopy Wide-field epifluorescence was performed on an Olympus IX83 inverted microscope with a ×100 objective, coupled to a Hamamatsu ORCA-Flash4.0 digital camera (Olympus Canada, Richmond Hill, Canada).

Techniques: Transfection, Epifluorescence Microscopy, Control

Journal: The Journal of Biological Chemistry

Article Title: mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

doi: 10.1074/jbc.RA118.002800

Figure Lengend Snippet: mTORC1 integrates multiple signals to control GSK3β nuclear localization. RPE cells were treated with either 100 μm A769662, 5 μm compound C, or 1 μm rapamycin, alone or in combination for 1 h (A); with 1 μm concanamycin for 1 h (B); or with amino acid-free EBSS medium for 2 h (C). D, RPE cells were transfected with siRNA targeting ULK1 or nontargeting siRNA (control). Following knockdown, RPE cells were treated with 1 μm rapamycin for 1 h and then were fixed and stained to detect endogenous GSK3β. Shown for each of these (left) are micrographs obtained by wide-field epifluorescence microscopy representative of three independent experiments. Scale bar, 20 μm. Also shown for each condition as GSK3β overlay are sample cellular and nuclear outlines and a box corresponding to a magnified image of a single cell. Also shown (right) is the mean GSK3β nuclear localization index ± S.E. (error bars) (n = 3 independent experiments, >30 cells/condition/experiment). *, p < 0.05 relative to the noninhibitor-treated condition (and in the control siRNA sample for D) of each experiment.

Article Snippet: Fluorescence microscopy Wide-field epifluorescence was performed on an Olympus IX83 inverted microscope with a ×100 objective, coupled to a Hamamatsu ORCA-Flash4.0 digital camera (Olympus Canada, Richmond Hill, Canada).

Techniques: Control, Transfection, Knockdown, Staining, Epifluorescence Microscopy

Journal: The Journal of Biological Chemistry

Article Title: mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

doi: 10.1074/jbc.RA118.002800

Figure Lengend Snippet: GSK3β Ser-9 phosphorylation is not required for GSK3β nuclear localization induced by inhibition of PI3K–Akt–mTORC1 signals. A, RPE cells were treated with either 10 μm LY294002, 5 μm Akti-1/2, or 1 μm rapamycin for 1 h. Shown are representative immunoblots of whole-cell lysates probed with anti-pSer-9 GSK3β or anti-total GSK3β antibodies. Also shown are mean anti-pSer-9 GSK3β levels (normalized to total GSK3β) ± S.E. (error bars) (n = 3); *, p < 0.05 relative to that in the control conditions (absence of LY294002, Akti1/2, and rapamycin). B, RPE cells were transfected with plasmids encoding HA-tagged WT or S9A GSK3β and then treated with 5 μm Akti-1/2 for 1 h, followed by detection of exogenous HA-GSK3β proteins. Shown (top) are micrographs obtained by wide-field epifluorescence microscopy representative of three independent experiments. Scale bar, 20 μm. Also shown for each condition as HA-GSK3β overlay are sample cellular and nuclear outlines and a box corresponding to a magnified image of a single cell. Also shown (bottom) is the mean HA-GSK3β nuclear localization index ± S.E. (n = 3, >30 cells/condition/experiment); *, p < 0.05 relative to control conditions (absence of Akti1/2 treatment). All Western blotting quantifications shown have been normalized to loading controls.

Article Snippet: Fluorescence microscopy Wide-field epifluorescence was performed on an Olympus IX83 inverted microscope with a ×100 objective, coupled to a Hamamatsu ORCA-Flash4.0 digital camera (Olympus Canada, Richmond Hill, Canada).

Techniques: Phospho-proteomics, Inhibition, Western Blot, Control, Transfection, Epifluorescence Microscopy

Journal: The Journal of Biological Chemistry

Article Title: mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

doi: 10.1074/jbc.RA118.002800

Figure Lengend Snippet: Rab7 controls GSK3β nuclear localization and GSK3β-dependent c-Myc levels. RPE cells were transfected with plasmids encoding dsRed-tagged WT or T22N Rab7 (A and C) or transfected with siRNA targeting APPL1 or nontargeting siRNA (control) (B). Some samples were then treated with 1 μm rapamycin for 1 h, followed by detection of endogenous GSK3β (A and B) or c-Myc (C). Shown (left panels) are micrographs obtained by wide-field epifluorescence microscopy representative of three independent experiments. Scale bar, 20 μm. Also shown for each condition as GSK3β overlay (A) or c-Myc overlay (B) are sample cellular and nuclear outlines and a box corresponding to a magnified image of a single cell. Also shown (right panels) is the mean ± S.E. (error bars) of the GSK3β nuclear localization index (A and B) (n = 3, >30 cells/condition/experiment) or total cellular c-Myc level (C) (n = 3, >30 cells/condition/experiment); *, p < 0.05 relative to control conditions (no rapamycin or CHIR99021 treatment).

Article Snippet: Fluorescence microscopy Wide-field epifluorescence was performed on an Olympus IX83 inverted microscope with a ×100 objective, coupled to a Hamamatsu ORCA-Flash4.0 digital camera (Olympus Canada, Richmond Hill, Canada).

Techniques: Transfection, Control, Epifluorescence Microscopy