ikkβ addgene  (Addgene inc)

Journal: Molecular cell

Article Title: α-Ketoglutarate-Activated NF-κB Signaling Promotes Compensatory Glucose Uptake and Brain Tumor Development.

doi: 10.1016/j.molcel.2019.07.007

Figure Lengend Snippet: Figure 6. a-KG-Activated NF-kB Promotes Tumor Cell Survival and Gliomagenesis (A and B) IKKb-depleted U87 or U251 cells reconstituted with rIKKb WT or R140L were treated with LG (0.5 mM) for 6 h (glucose and lactate assays; A) or for indicated time (cell viability assays; B). Data represent the mean ± SD of three independent experiments (**p < 0.01). (C–E) Luciferase-expressing, IKKb-depleted U87/EGFRvIII cells were reconstituted with rIKKb WT or R140L. IB analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments (C). These cells were intracranially injected into randomized nude mice (five mice per group). Representative images of bioluminescence imaging were presented, and luciferase intensities were quantified. Data represent the mean ± SD of luciferase intensity of five mice per group (D). Representative images of H&E-stained coronal brain sections or tumor boundaries were shown. Tumor volumes were measured. Data represent the mean ± SD of tumor volume of five mice per group (E).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human: U251 cells Cell library of the Chinese Academy of Sciences Cat# TCHu 58 Human: GSC11 cells Zhimin Lu lab; MD Anderson Cancer Center N/A Human: SK-Hep-1 cells Cell library of the Chinese Academy of Sciences Cat# TCHu109 Human: RKO cells Cell library of the Chinese Academy of Sciences Cat# TCHu116 Human: LN18 cells ATCC Cat# CRL-2610 Experimental Models: Organisms/Strains Mouse: BALB/c nude Lingchang Biotech N/A Oligonucleotides Control shRNA:ctcgcttgggcgagagtaa This paper N/A GDH1 shRNA:acttgctcatgtctgttca This paper N/A IKKb shRNA Addgene library Cat# 26209 RelA shRNA ThermoFisher Cat# TRCN0000014683 GLUT1 shRNA ThermoFisher Cat# TRCN0000043583 AMPK a1 shRNA:aaaatgagtctacagttat This paper N/A See Table S6 for Sequences of primers or response elements This paper N/A Recombinant DNA pCDH/hygro (+) 30SFB-GDH1 This paper N/A pCDH/hygro (+) -GDH1 This paper N/A pCDH/hygro (+) 30Flag-GDH1 This paper N/A pCMV-Flag-IKKb This paper N/A pCold I-GST-IKKb This paper N/A pCold I-His-GDH1 This paper N/A pCold I-His-IkBa This paper N/A pCDH/hygro (+)-50HA-RelA This paper N/A pCDH/hygro (+)-50HA-p100 This paper N/A pCDH/hygro (+)-RelA This paper N/A pCDH/hygro (+)-GLUT1 This paper N/A PGL3-TA- promoter-p53-Luc This paper N/A NF-kB-Luc reporter Fajian Hou Lab; University of Chinese Academy of Sciences N/A PGL3-TA-promoter-HIF1a-Luc This paper N/A PGL3-TA- promoter-c-Myc-Luc This paper N/A PGL3-Basic-GLUT1-Luc This paper N/A Software and Algorithms Graphpad Prism GraphPad https://www.graphpad.com Deposited Data RNA-Seq GEO database GSE121580 Raw image Mendeley data https://doi.org/10.17632/8t5rrg89hw.1

Techniques: Luciferase, Expressing, Injection, Imaging, Staining

Journal: Molecular cell

Article Title: α-Ketoglutarate-Activated NF-κB Signaling Promotes Compensatory Glucose Uptake and Brain Tumor Development.

doi: 10.1016/j.molcel.2019.07.007

Figure Lengend Snippet: Figure 7. GDH1 pS384 Correlates with the Malignancy and Prognosis of Human GBM (A) IHC analyses were performed with indicated antibodies in tumors from 58 GBM patients. Representative images of 3 tumors were shown. Correlation between GDH1 pS384 and nuclear RelA was analyzed (Pearson product moment correlation test). (B) Survival duration of 56 patients with low (29 cases, green curve) versus high (27 cases, red curve) GDH1 pS384 levels was compared (two-tailed log rank test). Landmark represents censored (alive at last clinical follow-up) patients. The table (top) shows the multivariate analysis (Cox regression). (C) IHC analyses were performed with anti-GDH1 pS384 in 40 diffuse astrocytoma tumors and 40 GBM tumors. GDH1 pS384 levels were compared between these two group (Student’s t test; two tailed; **p < 0.01). (D) Schematic model of the a-KG-activated NF-kB. Under low glucose, S384-phosphorylated GDH1 interacts with IKK complex. GDH1-produced a-KG directly activates IKKb and NF-kB signaling, which upregulates GLUT1 to promote glucose uptake and tumor cell survival under low glucose. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human: U251 cells Cell library of the Chinese Academy of Sciences Cat# TCHu 58 Human: GSC11 cells Zhimin Lu lab; MD Anderson Cancer Center N/A Human: SK-Hep-1 cells Cell library of the Chinese Academy of Sciences Cat# TCHu109 Human: RKO cells Cell library of the Chinese Academy of Sciences Cat# TCHu116 Human: LN18 cells ATCC Cat# CRL-2610 Experimental Models: Organisms/Strains Mouse: BALB/c nude Lingchang Biotech N/A Oligonucleotides Control shRNA:ctcgcttgggcgagagtaa This paper N/A GDH1 shRNA:acttgctcatgtctgttca This paper N/A IKKb shRNA Addgene library Cat# 26209 RelA shRNA ThermoFisher Cat# TRCN0000014683 GLUT1 shRNA ThermoFisher Cat# TRCN0000043583 AMPK a1 shRNA:aaaatgagtctacagttat This paper N/A See Table S6 for Sequences of primers or response elements This paper N/A Recombinant DNA pCDH/hygro (+) 30SFB-GDH1 This paper N/A pCDH/hygro (+) -GDH1 This paper N/A pCDH/hygro (+) 30Flag-GDH1 This paper N/A pCMV-Flag-IKKb This paper N/A pCold I-GST-IKKb This paper N/A pCold I-His-GDH1 This paper N/A pCold I-His-IkBa This paper N/A pCDH/hygro (+)-50HA-RelA This paper N/A pCDH/hygro (+)-50HA-p100 This paper N/A pCDH/hygro (+)-RelA This paper N/A pCDH/hygro (+)-GLUT1 This paper N/A PGL3-TA- promoter-p53-Luc This paper N/A NF-kB-Luc reporter Fajian Hou Lab; University of Chinese Academy of Sciences N/A PGL3-TA-promoter-HIF1a-Luc This paper N/A PGL3-TA- promoter-c-Myc-Luc This paper N/A PGL3-Basic-GLUT1-Luc This paper N/A Software and Algorithms Graphpad Prism GraphPad https://www.graphpad.com Deposited Data RNA-Seq GEO database GSE121580 Raw image Mendeley data https://doi.org/10.17632/8t5rrg89hw.1

Techniques: Two Tailed Test, Produced