Journal: STAR Protocols

Article Title: Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators

doi: 10.1016/j.xpro.2023.102762

Figure Lengend Snippet: Analysis of the preparation of two libraries (A) Representative of electrophoresis of first PCR product of Liu Human CRISPR Knockout Library with titered genome DNA as template. The backbone of this library is LentiCrispr V2. The best PCR condition is 4 μg genome DNA per reaction. (B) Representative of electrophoresis of first PCR product of Human CRISPR Metabolic Gene Knockout Library with titered genome DNA as template. The backbone of this library is LentiCrispr V1. The best PCR condition is 3 μg genome DNA per reaction. (C) Representative of electrophoresis of first and second PCR product of Liu Human CRISPR Knockout Library. For the second step PCR, primer pairs “i5 index N502” and “i7 index N701” were used. (D) Representative of electrophoresis of first and second PCR product of Human CRISPR Metabolic Gene Knockout Library. For the second step PCR, primer pairs “i5 index N502” and “i7 index N701” were used. (E) Upper panel: Representative bioanalyzer electropherograms of human metabolic libraries using an Agilent DNA 1000 kit. Lower panel: Representative bioanalyzer gel images of the same human metabolic libraries.

Article Snippet: Download the Human CRISPR Metabolic Gene Knockout Library file targets 2,981 human genes with 29,790 guide RNAs ( https://www.addgene.org/pooled-library/sabatini-human-crispr-metabolic-knockout/ ) as lentiCRISPR_v1_library.txt.

Techniques: Electrophoresis, CRISPR, Knock-Out, Gene Knockout

Journal: STAR Protocols

Article Title: Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators

doi: 10.1016/j.xpro.2023.102762

Figure Lengend Snippet:

Article Snippet: Download the Human CRISPR Metabolic Gene Knockout Library file targets 2,981 human genes with 29,790 guide RNAs ( https://www.addgene.org/pooled-library/sabatini-human-crispr-metabolic-knockout/ ) as lentiCRISPR_v1_library.txt.

Techniques: Recombinant, Modification, Saline, Staining, Viability Assay, DNA Purification, Purification, Gel Extraction, Plasmid Preparation, CRISPR, Knock-Out, Gene Knockout, Software, Cell Culture

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: IRS-1 immunohistochemistry performed on a tissue array from which 64 high-quality brain tumor biopsy specimens were selected a

Article Snippet: IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from ) (A) or the cytoplasm (case A5 from ) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens.

Techniques: Immunohistochemistry

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: Immunohistochemical detection of IRS-1. IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from Table 1) (A) or the cytoplasm (case A5 from Table 1) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens. IRS-1 nuclear structures were detected with anti-IRS-1 rabbit polyclonal antibody (catalog no. 06-248; Millipore) and FITC-conjugated anti-rabbit secondary antibodies. Nuclei were counterstained with DAPI (blue fluorescence). (C) Selected area adjacent to the tumor-infiltrating margin with a high number of tumor cells positive for IRS-1 nuclear structures (15 out of a total of 121 cells show IRS-1 nuclear structures). The percentage of tumor cells positive for IRS-1 nuclear structures was evaluated by using high-magnification confocal imaging (original magnification, ×100). At least 100 randomly selected fields per biopsy specimen were examined for 10 different glioblastoma biopsy specimens. (D) The same glioblastoma multiforme biopsy specimen immunolabeled with an irrelevant antibody (anti-BrdU primary antibody and FITC-conjugated secondary antibody). (E) Unaffected brain area, adjacent to the tumor depicted in panel C, immunolabeled with anti-IRS-1 rabbit polyclonal antibody.

Article Snippet: IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from ) (A) or the cytoplasm (case A5 from ) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens.

Techniques: Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Fluorescence, Imaging, Immunolabeling

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: IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from ) (A) or the cytoplasm (case A5 from ) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens.

Techniques: Injection, Formalin-fixed Paraffin-Embedded, Immunofluorescence, 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 ringlike structures in nuclei of cells expressing IRS-1 fused with a nuclear localization signal (NLS). (A and B) Confocal images of LN-229 human glioblastoma cells transiently transfected with the construct in which the CMV promoter drives the expression of human IRS-1 cDNA cloned in frame with the NLS and a myc tag (pALS1-NLS-IRS-1/mycTag). The unique nuclear ringlike structures were detected by either anti-IRS-1 rabbit polyclonal antibody (green fluorescence) (A) or anti-myc tag mouse monoclonal antibody (red fluorescence) (B), and the nuclei are labeled with DAPI (blue fluorescence). (C) Detection of IRS-1 ringlike structures by Nomarski contrast (arrows) in LN-229 cells expressing the pALS1-NLS-IRS-1/mycTag construct. In the same image, nucleoli are indicated by “no.” (D) Similar ringlike structures were also observed in another human glioblastoma cell line, U87MG; in normal human astrocytes (NHA); and in mouse embryo fibroblasts (MEF), all of which were transfected (transiently) with the same pALS1-NLS-IRS-1/mycTag plasmid vector.

Article Snippet: IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from ) (A) or the cytoplasm (case A5 from ) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens.

Techniques: Expressing, Transfection, Construct, Clone Assay, Fluorescence, Labeling, Plasmid Preparation

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: IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from ) (A) or the cytoplasm (case A5 from ) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens.

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

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: Accumulation of LC3 in the nucleus in cells positive for IRS-1 nuclear structures is associated with inhibition of autophagy. (A) Western blot analysis of nuclear extracts from HeLa/EV and HeLa/CL006 cells 0, 2, and 4 h following amino acid starvation. The last lane in the blot represents the cytosolic fraction from HeLa/CL006 cells without amino acid starvation (control). The blots were probed with anti-LC3 (Cell Signaling Technology) and anti-p62(SQSTM1) (Santa Cruz). Anti-histone H3 (Cell Signaling Technology) and anti-GAPDH (Santa Cruz Biotechnology) were used as nuclear and cytosolic markers, respectively. (B) Confocal images of HeLa cells cotransfected with the pALS-NLS-IRS-1/mycTag plasmid and the LC3-GFP baculovirus vector. IRS-1 nuclear structures (red fluorescence) were immunolabeled with mouse monoclonal anti-myc tag antibody (catalog number sc-40; Santa Cruz Biotechnology). Both nuclear LC3 and LC3 within autophagosomes are labeled with GFP. Colocalization between nuclear IRS-1 and nuclear LC3 is indicated by an arrow, and cells with autophagosomes are indicated by a white asterisk. Nuclei were counterstained with DAPI (blue fluorescence). (C) Drug resistance detected in tumor cells positive for IRS-1/LC3 nuclear structures. Temozolomide (TMZ)-sensitive LN-229 human glioblastoma cells were transfected with NLS–IRS-1–GFP(HA)/MitoRed and sorted to obtain three mixed populations, expressing low (L), medium (M), and high (H) levels of the transgenes (NLS–IRS-1–GFP and MitoRed). Resistance to temozolomide was tested in monolayer cultures in the presence of 10% FBS with or without 250 μM TMZ. LN-229 cells transfected with an empty vector were used as a control. In both panels, data represent average values ± standard deviations (n = 3). * indicates statistically significant differences between HeLa/EV and HeLa/CL006 cells or between LN-229/EV and LN229/NLS-IRS-1 cells; ** indicates statistically significant differences between LN-229/NLS-IRS-1(low) and LN-229/NLS-IRS-1(high) cells. The inset shows cell sorting and confocal analyses of LN-229 cells expressing the NLS–IRS-1–GFP(HA)/MitoRed transgene. Cells with low, medium, and high levels of the transgene (NLS–IRS-1–GFP) were selected and used for the TMZ resistance assay.

Article Snippet: IRS-1 was detected in formalin-fixed paraffin-embedded sections of glioblastoma tissues (GL803a tissue array; USBiomax, Inc.) by using anti-IRS-1 rabbit polyclonal (Millipore) and anti-rabbit HRP-conjugated secondary antibodies. (A and B) Two glioblastoma biopsy specimens in which IRS-1 is localized in either the nuclei of some tumor cells (case C2 from ) (A) or the cytoplasm (case A5 from ) (B). (C, F, and G) Examples of low-magnification (C) and high-magnification (F and G) confocal images of IRS-1 nuclear structures detected in a restricted number of cells from glioblastoma biopsy specimens.

Techniques: Inhibition, Western Blot, Plasmid Preparation, Fluorescence, Immunolabeling, Labeling, Transfection, Expressing, FACS