Journal: Neoplasia (New York, N.Y.)

Article Title: Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma 1

doi: 10.1016/j.neo.2018.08.007

Figure Lengend Snippet: Binding of NFIA, NFIB, NFIC, and NFIX to NFI binding sites in the HEY1 promoter. Nuclear extracts were prepared from U251 GBM cells transfected with control (pCH), NFIA (pCH-NFIA), NFIB (pCH-NFIB), NFIC (pCH-NFIC), or NFIX (pCH-NFIX) expression constructs. (A) Western blot analysis of transfected cells. Nuclear extracts (20 μg) were electrophoresed through an 8% polyacrylamide-SDS gel, electroblotted onto PVDF membranes, and immunostained with α-HA antibody or α-DDX1 antibody. (B) Electrophoretic mobility shift assays were performed with the indicated radiolabeled probes: −32 bp, −332 bp, −411 bp, and −794 bp. Probes were incubated with the indicated nuclear extracts (2 μg pCH, 3 μg NFIA, 4 μg NFIB, 1 μg NFIC, and 2 μg NFIX). Amounts of protein were adjusted to compensate for differences in expression of transfected HA-NFIs. DNA-protein complexes were electrophoresed through a 6% polyacrylamide gel buffered in 0.5× TBE.

Article Snippet: Total RNA was isolated from GBM cells using the RNeasy Plus Kit (Qiagen), and cDNA was synthesized with Superscript II reverse transcriptase (Life Technologies). qPCR was carried out using an ABI 7900HT Fast Real-Time PCR System, with gene-specific oligonucleotides labeled at the 5′ end with the fluorescent reporter dye FAM (NFIA, Hs00325656_m1; NFIB, Hs00232149_m1; NFIC, Hs00907819_m1; NFIX, Hs00958849_m1; GFAP, Hs00157674_m1; B-FABP, Hs00361426_m1; NES, Hs04187831_g1: HEY1, Hs01114113_m1; GAPDH, Hs99999905_m1) and Taqman Fast Master Mix (Life Technologies).

Techniques: Binding Assay, Transfection, Control, Expressing, Construct, Western Blot, SDS-Gel, Electrophoretic Mobility Shift Assay, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma 1

doi: 10.1016/j.neo.2018.08.007

Figure Lengend Snippet: Regulation of HEY1 promoter activity by NFI. U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs. Where indicated (2×), cells underwent two rounds of siRNA transfection. (A) NFIA , NFIB , NFIC , NFIX , and (B) HEY1 mRNA expression was analyzed by qPCR. GAPDH was used as an endogenous control. Similar data were obtained in two separate experiments. (C) U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs, followed 24 hours later by transfection with pGL3/HEY1. Cells were harvested 60 hours later, and luciferase activity was quantified. Changes in relative light units (RLU) are relative to RLU obtained in U251 GBM cells transfected with control (scrambled) siRNA and pGL3/HEY1. The data are from three experiments. SEM is indicated by error bars. Statistical significance, determined using the unpaired t test, is indicated by * ( P < .05) and ** ( P < .01).

Article Snippet: Total RNA was isolated from GBM cells using the RNeasy Plus Kit (Qiagen), and cDNA was synthesized with Superscript II reverse transcriptase (Life Technologies). qPCR was carried out using an ABI 7900HT Fast Real-Time PCR System, with gene-specific oligonucleotides labeled at the 5′ end with the fluorescent reporter dye FAM (NFIA, Hs00325656_m1; NFIB, Hs00232149_m1; NFIC, Hs00907819_m1; NFIX, Hs00958849_m1; GFAP, Hs00157674_m1; B-FABP, Hs00361426_m1; NES, Hs04187831_g1: HEY1, Hs01114113_m1; GAPDH, Hs99999905_m1) and Taqman Fast Master Mix (Life Technologies).

Techniques: Activity Assay, Transfection, Control, Expressing, Luciferase