Journal: Molecular and Cellular Biology

Article Title: Molecular and Structural Traits of Insulin Receptor Substrate 1/LC3 Nuclear Structures and Their Role in Autophagy Control and Tumor Cell Survival

doi: 10.1128/MCB.00608-17

Figure Lengend Snippet: Detection of IRS-1 nuclear structures in glioblastoma multiforme xenografts. Human GBM12 cells were injected into the brains of immunodeficient mice (1 × 105 cells in 2 μl of PBS) by using a Hamilton syringe and a stereotactic frame. The cells were allowed to form brain tumors for 2 weeks, and tumor-bearing mice were subsequently treated with aldoxorubicin (aldoxo) (24 mg/kg of body weight by tail vein injection) (23). Seven days after treatment, mice were sacrificed, and brain tumors were extracted, formalin fixed, paraffin embedded, and sectioned. (A to C) Immunofluorescence detection of IRS-1 (anti-IRS-1 rabbit polyclonal antibody, catalog no. 06-248; Millipore) in brain tumor sections from aldoxorubicin-treated (A) and control vehicle-treated (B) mice and in tumor-free brain tissue from an aldoxorubicin-treated mouse (C). (D) Frequency of tumor cells positive for IRS-1 nuclear structures. The images were evaluated by using high-magnification confocal microscopy (original magnification, ×100). Three tumors per group were evaluated, in which 100 randomly selected fields per tumor were examined (n = 3). Data represent average values ± standard deviations. (E) High-magnification image of a single tumor cell from an aldoxorubicin-treated mouse in which anti-IRS-1 antibody recognized the ringlike structure. The same cell is also visualized by Nomarski contrast, and nuclei are labeled with DAPI (blue fluorescence). The rectangle indicates an IRS-1-positive nuclear structure, and the arrow points to the three-dimensional reconstruction of the IRS-1 ringlike structure. The image was acquired by using an FV1000 confocal microscope (Olympus), and the 3-D surface reconstruction was generated by using SlideBook 5 software (Intelligent Imaging Innovations).

Article Snippet: To ensure the formation of large quantities of the IRS-1/LC3 nuclear structures, the attached cells were first starved with SFM (Dulbecco's modified Eagle's medium [DMEM] plus 0.1% BSA) for 72 h and subsequently stimulated with 10% FBS for 6 h. Once the nuclear structures were formed, live-cell time-lapse imaging was performed by using either a FluoView FV1000 confocal laser scanning microscope equipped with a live-cell stage-top incubation system (Pathology Devices, Inc., MD) for high-magnification, high-resolution, single-cell imaging or a VivaView LCV110U incubator microscope (Olympus) for low-magnification time-lapse imaging of a large number of cells.

Techniques: Injection, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Control, Confocal Microscopy, Labeling, Fluorescence, Microscopy, Generated, Software, Imaging

Journal: Molecular and Cellular Biology

Article Title: Molecular and Structural Traits of Insulin Receptor Substrate 1/LC3 Nuclear Structures and Their Role in Autophagy Control and Tumor Cell Survival

doi: 10.1128/MCB.00608-17

Figure Lengend Snippet: Detection of LC3 in IRS-1 ringlike structures. (A) Confocal images of two LN-229 human glioblastoma cells expressing the NLS–IRS-1–myc tag fusion protein (pALS1-NLS-IRS-1/mycTag). Double immunolabeling was performed using by anti-myc tag mouse monoclonal antibody (red) and anti-LC3 rabbit polyclonal antibody (green). DAPI labeling was used to visualize nuclei (blue). LC3 immunolabeling (green) is localized inside the ringlike structure immunolabeled with anti-myc tag antibody (red). (B) Single immunolabeling with anti-LC3 rabbit polyclonal antibody (red) was utilized to detect IRS-1/LC3 structures in HeLa cells stably expressing the NLS–IRS-1–GFP construct (see the legend to Fig. 4). To avoid potential spectral overlap (fluorescence bleedthrough), which in the case of the IRS-1–GFP nuclear rings can also excite the red channel, we used a secondary antibody conjugated with FarRed (Alexa Fluor 647). (C) Confocal images of IRS-1/LC3 nuclear structures found in glioblastoma biopsy specimens. Formalin-fixed, paraffin-embedded sections were immunolabeled with anti-IRS-1(pS612) mouse monoclonal (green) and anti-LC3 rabbit polyclonal (red) antibodies, and colocalization between IRS-1 and LC3 was evaluated by using confocal imaging in combination with SlideBook 5 Mask operation software. (D) Three-dimensional reconstruction of the IRS-1/LC3 nuclear ringlike structure from panel B. The image was acquired by using an Olympus FV1000 confocal microscope and processed by using SlideBook 5 software (3-D volume reconstruction).

Article Snippet: To ensure the formation of large quantities of the IRS-1/LC3 nuclear structures, the attached cells were first starved with SFM (Dulbecco's modified Eagle's medium [DMEM] plus 0.1% BSA) for 72 h and subsequently stimulated with 10% FBS for 6 h. Once the nuclear structures were formed, live-cell time-lapse imaging was performed by using either a FluoView FV1000 confocal laser scanning microscope equipped with a live-cell stage-top incubation system (Pathology Devices, Inc., MD) for high-magnification, high-resolution, single-cell imaging or a VivaView LCV110U incubator microscope (Olympus) for low-magnification time-lapse imaging of a large number of cells.

Techniques: Expressing, Immunolabeling, Labeling, Stable Transfection, Construct, Fluorescence, Formalin-fixed Paraffin-Embedded, Imaging, Software, Microscopy