Journal: The Journal of Clinical Investigation

Article Title: Isocitrate dehydrogenase mutations suppress STAT1 and CD8 + T cell accumulation in gliomas

doi: 10.1172/JCI90644

Figure Lengend Snippet: C57BL/6 mice received intracranial injections of 1 × 105 GL261 or SB28 glioma cells stably transfected with cDNA for either WT or the R132H form of IDH1. Day-21 tumors were removed from mice and further assessed. (A) RT-PCR analysis of SB28 tumor–derived RNA showing decreased expression of CTL response–related genes in SB28-MUT (MUT tumor; n = 4) versus SB28-WT (WT tumor; n = 4) tumors. (B) CXCL10 ELISA was performed on tumor-derived protein extracts from mice bearing GL261-WT or GL261-MUT tumors. (C) Representative fluorescence microscopy images from sections stained with DAPI (blue), CD3 (magenta), and CD8 (yellow) on frozen tumor sections from mice bearing GL261-WT or GL261-MUT tumors. Cells were imaged as described in Figure 2. Scale bars: 10 μm. (D) Quantification of CD3+CD8+ dp cells from GL261-WT (n = 14) and GL261-MUT (n = 16) tumors using StrataQuest software. P values were obtained using a 2-sided, unpaired t test.

Article Snippet: Cells were then transfected with 10 μg plasmids encoding cDNAs for either the IDH-WT (OriGene Technologies; RC210582) or the R132H IDH-MUT (OriGene Technologies; RC400096) with Lipofectamine 2000 and Opti-MEM (Thermo Fisher Scientific), according to the manufacturer’s protocol.

Techniques: Stable Transfection, Transfection, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Staining, Software

Journal: The Journal of Cell Biology

Article Title: CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

doi: 10.1083/jcb.201106138

Figure Lengend Snippet: CIIA physically interacts with SOS1. (A) HeLa cells were deprived of serum for 16 h, incubated without or with 100 ng/ml EGF for 5 min, lysed, and subjected to immunoprecipitation (IP) with the anti-CIIA antibody or with control preimmune IgG. The resulting precipitates as well as the lysates were subjected to immunoblotting (IB) with antibodies to SOS1 or CIIA. (B) 293T cells were transfected for 48 h with various combinations of expression vectors for the indicated proteins. Cell lysates were immunoprecipitated with the anti-Flag antibody, and the resulting precipitates were examined by immunoblot analysis with the anti-Myc antibody. Cell lysates were also immunoblotted with antibodies to Flag or to Myc. IgG H and IgG L indicate heavy and light chains, respectively, of IgG. (C and D) In vitro–translated 35 S-labeled SOS1 variants were pulled down with GST or GST-fused proteins immobilized on glutathione–agarose beads. The bead-bound 35 S-labeled proteins were analyzed by SDS-PAGE and autoradiography. The gels were also stained with Coomassie brilliant blue. A fraction (5%) of the 35 S-labeled protein input to the binding reaction is also shown. NT, N-terminal domain; CEN, central domain; CT, C-terminal domain.

Article Snippet: 35 S-labeled SOS1 and deletion mutants thereof were produced in vitro with the use of the reticulocyte lysate system (T n T; Promega).

Techniques: Incubation, Immunoprecipitation, Western Blot, Transfection, Expressing, In Vitro, Labeling, SDS Page, Autoradiography, Staining, Binding Assay

Journal: The Journal of Cell Biology

Article Title: CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

doi: 10.1083/jcb.201106138

Figure Lengend Snippet: CIIA regulates EGF-induced Ras and Erk activation. (A) MDCK/control or MDCK/CIIA-Flag cells were deprived of serum for 16 h and incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were immunoprecipitated (IP) with anti-SOS1 antibody, and the pellets were immunoblotted (IB) with anti-Ras antibody. Cell lysates were also examined directly by immunoblotting with antibodies to SOS1, Ras, or Flag. (B and C) MDCK/CIIA-Flag and MDCK/control cells (B) or HeLa cells stably expressing either GFP (control) or CIIA siRNA (C) were deprived of serum for 16 h and then incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were subjected to pull-down (PD) with GST-RBD and assayed for Ras activity.

Article Snippet: 35 S-labeled SOS1 and deletion mutants thereof were produced in vitro with the use of the reticulocyte lysate system (T n T; Promega).

Techniques: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Activity Assay

Journal: The Journal of Cell Biology

Article Title: CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

doi: 10.1083/jcb.201106138

Figure Lengend Snippet: CIIA promotes EGF-induced SOS1–Rac1 signaling. (A) HeLa cells transfected with GFP or SOS1 siRNA for 12 h were deprived of serum for 16 h and then incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were pulled down (PD) with GST-CRIB and assayed for Rac1 activity. (B) 293T cells were transfected for 48 h with vectors for the indicated proteins, after which cell lysates were subjected to a pull-down assay with GST-CRIB as in A. (C) The GEF activity of SOS1-DHPH on Rac1 was determined in vitro in the absence or presence of CIIA by fluorescence spectroscopy with the use of the GEF exchange assay kit as described in Materials and methods. (top) Relative fluorescence intensity data from one representative experiment. (bottom) Data are the means ± SD of three independent experiments. Results are expressed as the mant-GTP incorporation into GST-Rac1 after 480 s relative to time 0 after adding His 6 -SOS1-DHPH. *, P < 0.05. (D) MDCK/CIIA-Flag and MDCK/control cells were deprived of serum for 16 h and incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were subjected to Rac1 activity assay as in A. (E) HeLa cells were transfected for 24 h with the indicated combinations of GFP, CIIA, or E3B1 siRNA and a vector encoding CIIA*-Myc. Then, the cells were deprived of serum for 16 h and incubated for 5 min in the presence or absence of 100 ng/ml EGF. Cell lysates were assayed for Rac1 activity as in A. (F) HeLa-siGFP or HeLa-siCIIA cells were deprived of serum for 16 h and then incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were examined by immunoblotting (IB) with antibodies to phospho(Thr 423 )-PAK1, PAK1, or CIIA.

Article Snippet: 35 S-labeled SOS1 and deletion mutants thereof were produced in vitro with the use of the reticulocyte lysate system (T n T; Promega).

Techniques: Transfection, Incubation, Activity Assay, Pull Down Assay, In Vitro, Fluorescence, Spectroscopy, Plasmid Preparation, Western Blot

Journal: The Journal of Cell Biology

Article Title: CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

doi: 10.1083/jcb.201106138

Figure Lengend Snippet: CIIA promotes SOS1-mediated activation of Rac1. (A and B) MDCK/CIIA-Flag and MDCK/control cells (A) or HeLa cells expressing GFP (control) or CIIA siRNA (B) were deprived of serum for 16 h, incubated for 5 min with or without 100 ng/ml EGF, and lysed. Cell lysates were immunoprecipitated (IP) with the anti-SOS1 antibody, and the resulting precipitates were immunoblotted (IB) with the anti-EPS8 antibody. (C) 293T cells were transfected for 48 h with vectors for EPS8-Myc, HA-SOS1, and CIIA-Flag as indicated. Cell lysates were immunoprecipitated with the anti-HA antibody, and the precipitates were immunoblotted with the anti-Myc antibody. (D) HeLa cells were transfected with either GFP or E3B1 siRNA alone or together with a vector encoding CIIA-Flag. After 8 h of transfection, the cells were deprived of serum for 16 h and then incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were immunoprecipitated with the anti-SOS1 antibody, and the resulting precipitates were immunoblotted with the anti-EPS8 antibody. Cell lysates were also subjected to pull-down (PD) with GST-CRIB and assayed for Rac1 activity. (E) HeLa cells stably expressing GFP or CIIA siRNA were deprived of serum for 16 h, transferred to the upper chambers of a Transwell plate, incubated for 24 h with (black bars) or without (white bars) 100 ng/ml EGF in the lower chambers, and analyzed for migration. (F) HeLa cells stably expressing GFP or CIIA siRNA were serum starved for 16 h, incubated for 5 min with (black bars) or without (white bars) 100 ng/ml EGF, and stained with Alexa Fluor red–conjugated phalloidin to detect F-actin. Arrowheads indicate F-actin–enriched membrane ruffles. The number of cells with ruffles were counted and expressed as the percentages relative to the total number of cells. Data are means ± SD from three independent experiments. *, P < 0.01. Bar, 10 µm.

Article Snippet: 35 S-labeled SOS1 and deletion mutants thereof were produced in vitro with the use of the reticulocyte lysate system (T n T; Promega).

Techniques: Activation Assay, Expressing, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Activity Assay, Stable Transfection, Migration, Staining

Journal: The Journal of Cell Biology

Article Title: CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

doi: 10.1083/jcb.201106138

Figure Lengend Snippet: CIIA mediates TGF-β–induced migration of A549 cells. (A, B, and G) A549 cells were transfected either with GFP (control), SOS1 (A), or CIIA (G) siRNA oligonucleotides or with a vector for Rac1N17 or the corresponding empty vector (B). After 8 h of transfection, the cells were deprived of serum for 16 h, transferred to the upper chambers of a Transwell plate, incubated for 48 h with (black bars) or without (white bars) 2 ng/ml TGF-β in the lower chambers, and analyzed for migration. Data are means ± SD from three independent experiments. *, P < 0.01. (C) A549 cells were transfected with GFP or SOS1 siRNA for 16 h, deprived of serum for 10 h, and incubated with or without 2 ng/ml TGF-β for 16 h. Cell lysates were then subjected to pull-down (PD) with GST-CRIB and assayed for Rac1 activity. (D) A549 cells were incubated with or without 10 µM BMS345541 for 1 h and then in the presence or absence of 2 ng/ml TGF-β for 6 h (for quantitative RT-PCR analysis of CIIA mRNA) or for 16 h (for immunoblot [IB] with antibodies to CIIA or α-tubulin [loading control]). RT-PCR data are means ± SD of triplicates from a representative experiment. (E) A549 cells were left untreated or treated with 2 ng/ml TGF-β for 16 h, lysed, and immunoprecipitated (IP) with the anti-CIIA antibody or rabbit preimmune IgG. The resulting precipitates were immunoblotted with the anti-SOS1 antibody. (F) A549 cells were transfected for 12 h with GFP or CIIA siRNA oligonucleotides, deprived of serum for 16 h, and incubated with or without 2 ng/ml TGF-β for 16 h. Cell lysates were then subjected to Rac1 activity assay as in C. Cell lysates were also immunoprecipitated with the anti-SOS1 antibody, and the resulting precipitates were immunoblotted with the anti-EPS8 antibody.

Article Snippet: 35 S-labeled SOS1 and deletion mutants thereof were produced in vitro with the use of the reticulocyte lysate system (T n T; Promega).

Techniques: Migration, Transfection, Plasmid Preparation, Incubation, Activity Assay, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation