rnascope probe mm-lrig1  (Advanced Cell Diagnostics Inc)

Journal: Oncogenesis

Article Title: Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

doi: 10.1038/s41389-017-0012-8

Figure Lengend Snippet: Newborn Ntv-a mice were transduced with PDGFB-expressing avian retroviruses. At 12 weeks of age, the mice were sacrificed, and their brains were dissected and analyzed via in situ hybridization. a Micrographs showing images of in situ hybridized sections of normal brain (upper row), grade II glioma (middle row), and grade III–IV glioma (lower row). The left panel shows sections hybridized with the negative control probe, whereas the right panel shows sections hybridized with the Lrig1 probe. Scale bar, 100 μm. b Quantification of the total number of signals (single spots and clusters) per area in normal brain ( n = 3), grade II glioma ( n = 3), and grade III–IV glioma ( n = 3). There was no significant difference in Lrig1 expression (in situ hybridization signals per area) among the groups

Article Snippet: An RNAScope probe set targeting Lrig1 (probe-Mm-Lrig1, Mouse: 310521) was custom designed by and purchased from Advanced Cell Diagnostics.

Techniques: Transduction, Expressing, In Situ Hybridization, In Situ, Negative Control

Journal: Oncogenesis

Article Title: Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

doi: 10.1038/s41389-017-0012-8

Figure Lengend Snippet: a Schematic representation of wild-type, conditional knockout, and disrupted Lrig1 alleles. A PKG-neo selection cassette was inserted downstream of exon 1 of Lrig1 . Both exon 1 and the PKG-neo cassette were flanked by loxP sites; thus, both regions could be deleted in a single step by mating the mice with OzCre mice to generate the disrupted Lrig1 allele. b Relative Lrig1 mRNA expression levels in mouse brains from wild-type (WT), Lrig1 -heterozygous (HZ), and Lrig1 -deficient (KO) mice. Total RNA was prepared from the brains of 1-week-old mice, and the Lrig1 expression levels were determined with quantitative RT-PCR. The Lrig1 levels were normalized to those of the internal control gene Rn18s . An arbitrary scale was established such that the mean Lrig1 levels in the mouse reference RNA had a value 1. In the Lrig1 -deficient mice, Lrig1 could not be detected in any of the samples analyzed. Error bars indicate ±standard deviations. † p = 6.6 × 10 −7 , Student’s t -test. c Western blot analysis of brain lysates from 1-week-old wild-type (WT), Lrig1 -heterozygous (HZ), and Lrig1 -deficient (KO) mice using antibodies against Lrig1 or actin. d Quantification of Western blots of brain lysates from wild-type mice, Lrig1 -heterozygous mice, and Lrig1 -knockout mice. The mean Lrig1/actin ratios are shown on an arbitrary scale. Error bars indicate ±standard deviations. * p = 0.025, ** p = 0.0045, and *** p = 0.00088, Student’s t -test

Article Snippet: An RNAScope probe set targeting Lrig1 (probe-Mm-Lrig1, Mouse: 310521) was custom designed by and purchased from Advanced Cell Diagnostics.

Techniques: Knock-Out, Selection, Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Oncogenesis

Article Title: Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

doi: 10.1038/s41389-017-0012-8

Figure Lengend Snippet: Newborn mice with different Lrig1 genotypes were transduced with PDGFB-encoding RCAS retroviruses. At 12 weeks of age, the mice were sacrificed, and their brains were examined. Circles show no tumors (light gray), low-grade tumors (grade II, intermediate gray), and high-grade tumors (grade III–IV, dark gray) according to Lrig1 genotype; wild-type ( Lrig1+/+ , n = 61), Lrig1 -heterozygous ( Lrig1+/− , n = 113), and Lrig1 -deficient ( Lrig1−/− , n = 41). The Lrig1 -heterozygous mice developed significantly more high-grade tumors than did wild-type mice (Pearson’s χ 2 test, p = 0.002)

Article Snippet: An RNAScope probe set targeting Lrig1 (probe-Mm-Lrig1, Mouse: 310521) was custom designed by and purchased from Advanced Cell Diagnostics.

Techniques: Transduction

Journal: Oncogenesis

Article Title: Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

doi: 10.1038/s41389-017-0012-8

Figure Lengend Snippet: TB101 and TB107 cells were transduced with either control vector, to produce TB101 Puro and TB107 Puro cells or a stable LRIG1 expression vector to produce TB101 LRIG1 and TB107 LRIG1 cells. a Nude mice were intracranially injected with TB107 Puro or TB107 LRIG1 cells. Four months after injection, the mice were killed, and their brains were analyzed using immunohistochemistry. Micrographs showing representative images of mouse brains transplanted with TB107 Puro cells or TB107 LRIG1 cells and stained for human vimentin to visualize the tumor cells. b Representation showing the quantification of the number of satellite tumors per section from mice with TB107 Puro ( n = 10) and TB107 LRIG1 tumors ( n = 12). Horizontal lines indicate the medians of the respective group. The difference in the number of satellite tumors surrounding the TB107 Puro and TB107 LRIG1 primary tumors was significant (Student’s t -test, p = 0.02). c Proliferation rates of the respective cell lines. Error bars indicate ± standard deviations of the means from triplicate samples. One of three similar experiments is shown. The difference in proliferation rate between TB101 Puro and TB101 LRIG1 cells was significant (** p < 0.01, Student’s t -test). d Migration of the respective cell lines. Cells were seeded in transwell inserts with serum-free medium in the upper chamber and medium containing 5% FBS in the lower chamber. After the cells were incubated 24 h, the inserts were fixed and stained with crystal violet, and the cells that migrated across the membrane were counted. The mean number of migratory cells per ten high-power fields in one of three similar experiments is shown; standard deviations of triplicate samples are indicated with error bars. *** p < 0.001, Student’s t -test

Article Snippet: An RNAScope probe set targeting Lrig1 (probe-Mm-Lrig1, Mouse: 310521) was custom designed by and purchased from Advanced Cell Diagnostics.

Techniques: Transduction, Control, Plasmid Preparation, Expressing, Injection, Immunohistochemistry, Staining, Migration, Incubation, Membrane

Journal: Oncogenesis

Article Title: Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

doi: 10.1038/s41389-017-0012-8

Figure Lengend Snippet: a TB101 DoxLRIG1 (left column) and TB107 DoxLRIG1 (right column) cells with a doxycycline-inducible LRIG1 allele were incubated in the presence or absence of doxycycline for 48 h, after which the cells were transferred to transwell inserts and incubated in the presence or absence of doxycycline for an additional 48 h. Micrographs showing representative images of migratory cells incubated in the presence (Dox+) or absence (Dox−) of doxycycline. b Bar charts showing the quantifications of the number of migratory cells per five high-power microscopy fields. One of three independent experiments is shown. In each experiment, triplicate wells were analyzed for each treatment, and the error bars indicate standard deviations. * p < 0.05, Student’s t -test. c Immunoblots showing the induction of LRIG1 by doxycycline at various time points; actin is shown as the loading control. d TB101 DoxLRIG1 (left panel) or TB107 DoxLRIG1 (right panel) cells were incubated in the presence (Dox+) or absence (Dox−) of doxycycline for 48 h followed by lysis, and the phospho-RTK levels were analyzed using a phospho-RTK array. The positions and identities of detectable spots are indicated with arrows and include p-EGFR, p-MET, p-INSR, p-IGF1R, p-AXL, p-RET and p-VEGFR2. e TB107 DoxLRIG1 cells were incubated in the presence or absence of doxycycline for the indicated times followed by lysis, and an immunoblot analysis for LRIG1, p-EGFR (Tyr1086), total EGFR, p-MET (Tyr1234/1235), and total MET was conducted with actin as the loading control. f TB107 DoxLRIG1 cells were incubated in the presence or absence of doxycycline for 48 h followed by lysis, and total RET and p-RET (panTYR) were analyzed using specific ELISA kits. One of three similar experiments depicting the mean of triplicate samples is shown; standard deviations are indicated by error bars. g–i Pharmacologic and LRIG1-induced suppression of TB107 cell migration. LRIG1 expression was induced by culturing TB107 DoxLRIG1 cells with doxycycline for 48 h. Then, the cells were seeded in transwell inserts in the presence or absence of the MET inhibitor PHA-665752 for 24 h. To enhance transwell migration via chemotaxis, the medium in the upper chamber was serum-free, whereas the medium in the lower chamber contained 5% FBS. g At the end of the experiment, the transwell inserts were fixed and stained with crystal violet, and the number of migratory cells was counted. The mean number of migratory cells per ten high-power fields of three wells from one of three independent experiments is shown. Standard deviations of triplicate wells are indicated by error bars. * p < 0.05 and ** p < 0.01, Student’s t -test. h In parallel experiments, cells were seeded in six-well plates but were otherwise treated in the same manner as the cells in the transwell chambers. After 12 or 24 h of incubation, the cells were lysed and subjected to immunoblot analyses for LRIG1, p-EGFR (Tyr1086), total EGFR, p-MET (Tyr1234/1235), and total MET with actin as the loading control. i Quantification of the p-MET/actin ratios 12 h (left) and 24 h (right) after the induction of LRIG1 . The mean ratios of three independent experiments are shown with standard deviations indicated by error bars. * p < 0.05, ** p < 0.01, and *** p < 0.001, Student’s t -test

Article Snippet: An RNAScope probe set targeting Lrig1 (probe-Mm-Lrig1, Mouse: 310521) was custom designed by and purchased from Advanced Cell Diagnostics.

Techniques: Incubation, Microscopy, Western Blot, Control, Lysis, Enzyme-linked Immunosorbent Assay, Migration, Expressing, Chemotaxis Assay, Staining