Journal: PLoS ONE

Article Title: Induction of interferon-β and interferon signaling by TRAIL and Smac mimetics via caspase-8 in breast cancer cells

doi: 10.1371/journal.pone.0248175

Figure Lengend Snippet: MCF-7 cells were treated with LCL161 (10 μM) and/or TRAIL (100 ng/mL) for indicated time periods and total cell lysates were analyzed by immunoblotting for cIAP1 (A) the NFKB2 gene product p100 and p52 (B) or phosphorylated and total STAT1 (D). Intensity of bands in B and D were quantified (C and E) and normalized to LCL161+TRAIL (C) or TRAIL (E) treatment for 48 h. Protein levels of cIAP1 (F), p100 and p52 (G), or phosphorylated and total STAT1 (H) were measured in CAMA-1 cells that had been treated with LCL161 and/or TRAIL for 8 h in the absence or presence of zVAD-FMK (20 μM). Figures are representative of three independent experiments. Data in C and E indicate individual data points with bars representing the mean and error bars representing SEM, n = 3. * denotes p<0.05, ** p<0.01, *** p<0.001 using ANOVA followed by Tukey’s honest significance test.

Article Snippet: This was followed by incubation with primary antibodies against phosphorylated STAT1 (1:400, #9167), total STAT1 (1:400, #9172), FADD (1:400, #27825), caspase-8 (1:300, #9746), and FLIP (1:400, #56343) all from Cell Signaling Technology; p100/p52 (1:300, ab131539) and IFNAR1 (1:400, ab124764) from Abcam; cIAP1 (1:200, AF8181, R&D Systems); actin (1:2,000, #0869100, MP Biomedicals); and RIP1 (1:500, #610458, BD Biosciences).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: BIRC3 expression segregates mesenchymal GBM from the other GBM subtypes. Normalized log2 expression was compared across the neural, proneural, classical, and mesenchymal GBM subtypes. Results are represented in box plot format: ( A ) BIRC3 ; ( B ). BIRC2 ; ( C ) BIRC5 ; ( D ) NF1; ( E ) ZEB1 ; and ( F ) CREB1 .

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Expressing

Journal: Scientific Reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: BIRC3 expression is a unique identifier of mesenchymal GBM. Normalized log2 expression of BIRC3 was plotted against other proposed mesenchmymal-selective genes in a scatter plot, colored by GBM subtypes and with r representing Pearsons correlation coefficient. ( A ) BIRC3 versus CREB1 expression. ( B ) BIRC3 versus NF1 expression. ( C ) BIRC3 versus ZEB1 expression.

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Expressing

Journal: Scientific Reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: BIRC3 is expressed at higher level in the tumor cell niche compared to the vascular niche in GBM. Human GBM tissue microarray was stained for BIRC3 (Brown). BIRC3 expression can be compared between GBM tumor cell niche and vascular endothelial cell niche ( A,B ). Please note the focus of microvascular proliferation which shows negligible BIRC3 expression (Red arrow).

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Microarray, Staining, Expressing

Journal: Scientific Reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Hypoxia induces BIRC3 expression in GBM ex vivo and in vivo . ( A ) U87 GBM cells were cultured under hypoxic conditions (1% O 2 ) for 24 hr and BIRC3 gene expression was analyzed by RT-PCR. Data are representative of three independent experiments (p < 0.05). ( B ) U87 cells were cultured under hypoxia conditions (1% O 2 ) for the indicated intervals and BIRC3 protein levels were determined by western blot. Data are representative of three independent experiments. Whole images for Western-blot can be found in the Supplementary Figure . ( C – E ) GBM xenografts were established by by injecting 2 × 10 6 U87 cells on the flank of 6–8 week nude mice. At 6 weeks, mice were sacrificed and xenografts were assessed for BIRC3 (brown) and CA9 (pink) or HIF-1α (pink) expression by IHC. ( C ) BIRC3 and CA9 expression. ( D ) HIF-1α expression. ( E ) BIRC3 and HIF-1α expression.

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Expressing, Ex Vivo, In Vivo, Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Inhibition of HIF-1α blocks hypoxia-induced up-regulation of BIRC3 expression in GBM. ( A,B ) U87 GBM cells, or these cells transfected with HIF-1α siRNA for 48 hr, were cultured under hypoxia conditions (1% O 2 ) for 24 hr. Efficiency of HIF-1α knockdown and effects on BIRC3 gene expression were determined by qRT-PCR (n = 3 independent experiments, p < 0.05). (C) BIRC3 protein level were also assessed following knockdown of HIF-1α + /− hypoxia (n = 3 independent experiments). Whole images for Western-blot can be found in the Supplementary Figure . ( D ) ChIP of HIF-1α on the BIRC3 gene promoter was performed in U87 GBM cells exposed to hypoxia (1% O 2 for 24 hr).

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Inhibition, Expressing, Transfection, Cell Culture, Knockdown, Gene Expression, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: BIRC3 silencing impairs hypoxia-induced survival of GBM to radiotherapy (RT). 1 × 10 4 U87 MG GBM cells were cultured in 96 well plate under hypoxia (1% O 2 ) condition for 12 hr and irradiated with 4 Gy. Cells were returned to hypoxia conditions for another 12 hr and harvested. BIRC3 mRNA and protein expression were analyzed by RT-PCR ( A ) and Western blot, respectively ( B ). Similar results were obtained from three independent experiments (p < 0.05). Whole images for Western-blot can be found in the Supplementary Figure . ( C ) U87 GBM cells with or without BIRC3 siRNA pretreatment (48 hr earlier) were cultured under hypoxia (1% O 2 ) for 12 hr and irradiated with 2 Gy, 4 Gy, 6 Gy or 8 Gy. Cells were returned to hypoxia conditions for another 24 hr and cell survival were assessed using an XTT Cell Viability Assay Kit. Data are representative of three independent experiments (p < 0.05).

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Cell Culture, Irradiation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Viability Assay