Journal: The Journal of Neuroscience

Article Title: Developmental Regulation of Sensory Neurite Growth by the Tumor Necrosis Factor Superfamily Member LIGHT

doi: 10.1523/JNEUROSCI.3566-08.2009

Figure Lengend Snippet: LIGHT, HVEM, and LTβR are differentially expressed in the developing nervous system. A, Quantification of the relative levels of LIGHT, HVEM, and LTβR mRNAs in total RNA extracted from nodose ganglia (ND), superior cervical ganglia (SCG), trigeminal ganglia (TG), hippocampus (HP), and cortex (CX) of P0 mice by real-time PCR. The levels of these mRNAs were calculated relative to GAPDH mRNA and normalized to a value of 1.0 for the nodose data. Means ± SEM of assays performed on RNA extracted from three separate sets of tissue, (*p < 0.05 and **p < 0.01 indicate statistically significant difference between the indicated bar and all other bars). B, Immunohistochemical localization and relative levels of expression of LIGHT, HVEM, and LTβR in sections of P0 nodose ganglia, vagus nerve, and SCG. Scale bars, 50 μm. C, Photomicrographs of representative fields of dissociated cultures of P0 nodose neurons that were cultured for 24 h in medium containing 10 ng/ml BDNF before being triple labeled with either anti-β-III tubulin, anti-LIGHT, and DAPI (top row), anti-β-III tubulin, anti-HVEM, and DAPI (second row), or anti-β-III tubulin, anti-LTβR, and DAPI (third row). The lower row shows a representative nodose neuron cultured from a P0 LIGHT-deficient mouse stained for anti-β-III tubulin, anti-LIGHT, and DAPI. Scale bar, 50 μm. D, Quantification of the relative levels of LIGHT, HVEM, and LTβR mRNAs in total RNA extracted from nodose ganglia of E15, E18, P0, and P5 mice by real-time PCR. The levels of these mRNAs were calculated relative to GAPDH mRNA and normalized to a value of 1.0 at the P0 age point (mean ± SEM of assays performed on RNA extracted from three separate sets of ganglia at each age).

Article Snippet: The cells were incubated overnight at 4°C with primary antibody in 1% BSA (anti-β-III tubulin, 1:1000, Promega; anti-LIGHT, anti-HVEM and anti-LTβR, 1:50 Santa Cruz).

Techniques: Real-time Polymerase Chain Reaction, Immunohistochemical staining, Expressing, Cell Culture, Labeling, Staining

Journal: Immunity, Inflammation and Disease

Article Title: Tumor Cell‐Expressed Herpesvirus Entry Mediator Regulates Proliferation and Adaptive Immunity in Ovarian Cancer

doi: 10.1002/iid3.70175

Figure Lengend Snippet: Knockdown of Tnfrsf14 inhibited OvCa cell proliferation. (A) The murine OvCa cell line ID8 was infected with lentivirus to generate stable Tnfrsf14 KD cells. The shRNA‐mediated Tnfrsf14 knockdown efficiency was detected by western blot analysis. Ctrl ‐ID8 cells: sh ‐ Ctrl , Tnfrsf14 KD ‐ID8 cells: sh ‐ Tnfrsf14 . (B) The CCK−8 assay was used to determine the difference in cell viability between Ctrl ‐ID8 and Tnfrsf14 KD ‐ID8 cells. (C) The colony formation assay was used to detect the proliferation ability of Ctrl ‐ID8 and Tnfrsf14 KD ‐ID8 cells. The representative digital images (left) and statistical analysis (right) were shown. (D) The protein levels of XBP1s (the spliced form of XBP1), XBP1u (the unspliced form of XBP1), and c‐Myc in Ctrl ‐ID8 and Tnfrsf14 KD ‐ID8 cells were performed by western blot analysis. (E) The Ctrl ‐ID8 and Tnfrsf14 KD ‐ID8 cells were treated with 50 μΜ IXA4 or equal volumes of DMSO for 4 h and the protein levels of XBP1s, XBP1u, and c‐Myc were performed by western blot analysis. (F) The Ctrl ‐ID8 and Tnfrsf14 KD ‐ID8 cells were plated and treated with 50 μΜ IXA4 or equal volumes of DMSO for 4 h. The EdU assay was used to detect the difference in cell proliferation among each group. The representative digital images were shown. Scale bar: 200 μm. (G) The statistical analysis of the ratio of EdU‐positive cells among each group was shown. (H) The mRNA expression level of XBP1 in specimens from patients with benign tumors ( n = 7) and OvCa ( n = 26) was detected by quantitative RT‐PCR. Data were presented as means ± SD. Two‐tailed unpaired Student's t ‐test in (B), (C), (G), and (H). * p < 0.05, ** p < 0.01, *** p < 0.001. CCK‐8, Cell Counting Kit‐8; DMSO, dimethyl sulfoxide; EdU, 5‐ethynyl‐2′‐deoxyuridine; ns, no significance; OvCa, ovarian cancer; RT‐PCR, real‐time polymerase chain reaction; SD, standard deviation; shRNA, short hairpin RNA; TNFRSF14, tumor necrosis factor superfamily member 14.

Article Snippet: The primary antibodies used were as follows: HVEM (10138‐1‐AP; Proteintech, Chicago, USA), XBP1 (220783; Abcam, Cambridge, UK), c‐Myc (18583; CST, Danvers, USA), phospho‐NF‐κB p65‐Ser536 (3033T; CST, Danvers, USA), phospho‐STAT1‐Y701 (AP0135; ABclonal, Woburn, USA), phospho‐STAT5‐Y694 (AP0887; ABclonal, Woburn, USA), phospho‐STAT6‐Y641 (ab235591; Abcam, Cambridge, UK), and β‐Actin (AC026; ABclonal, Woburn, USA).

Techniques: Knockdown, Infection, shRNA, Western Blot, CCK-8 Assay, Colony Assay, EdU Assay, Expressing, Quantitative RT-PCR, Two Tailed Test, Cell Counting, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation