Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 1: PDL1 expression in clinical brain tumor samples, glioma cell lines and CSF samples from the immunocompetent glioma mice model. A) Graph (left) represents PDL1 mRNA expression in brain tumors and melanomas (cBioPortal (http://www.cbioportal.org)). Note that the average PDL1 mRNA level in GBM is similar to the average PDL1 mRNA level in melanomas. The PDL1 values in GBM and low-grade gliomas which exceed the average PDL1 mRNA level in melanomas are highlighted in the red box. Kaplan-Meier plot (right) illustrates survival rates of glioma patients with high and low PDL1 expression levels; 9% versus 24% of 2 year survival for high and low PDL1 expression; the difference is significant, P=0.02 (data has been obtained from the Human Pathology Atlas). B) Immunohistochemical detection of PDL1 in the tissue microarray of normal and brain tumor samples. The images were taken at 40x magnification. C) Western blot illustrates PDL1 and Actin protein levels in control and brain tumor clinical samples. Graph represents PDL1 to Actin ratios for corresponding protein samples; 0.87 ± 0.32 (n=7) versus 0.19 ± 0.1 (n=7) for tumor and normal samples, respectively, the difference is significant, P=0.003. Two samples (marked by light grey color) of patients with hemorrhage have been excluded from the average. D) Western blots illustrate PDL1 protein levels in established and PDGx glioma cell lines (left) and in the extracellular media collected from these cell lines (right). The protein content of the collected media was concentrated six folds before an analysis (see method). The graph (right) provides PDL1 concentrations in CSF samples from control and tumor-bearing mice (see method). The immunocompetent glioma mice model with GL261 cells was utilized for this experiment. E) Images illustrate interactions of tumor neuro spheres from XD456 (a) and from two parental U251-IDH1-R132H cell lines with different PDL1 expression levels (b) with primary T-cells. Note that tumor neuro spheres with high PDL1 levels keep their integrity after 48 hours of interaction with primary T-cells (illustrated in the insert). The graph represents the average percent of neuro spheres after 48 hours of interaction with T-cells, 89 ± 7% (n=4) and 14 ± 5% (n=4) for cell lines with high and low PDL1 levels, respectively. The difference is significant, P=0.0002. Primary T-cells were loaded with calcein, AM (green) before experiments for visualization.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Microarray, Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 2: HIF1A accumulation evokes PDL1 up-regulation in glioma cell lines. A) HIF1A accumulations induced by CoCl2 treatment of U251 and U87 cell lines evoke PDL1 up-regulation in protein and mRNA levels. Western blot illustrates HIF1A and PDL1 levels in nuclear and cytoplasmic fractions, respectively, in control and after treatment with CoCl2 (85uM). Lamin A/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. Graphs illustrate normalized PDL1/ GAPDH mRNA ratios after cell treatment with CoCl2 (85uM) at different time points. In each experiment, data has been normalized to the corresponding ratios in untreated cells, results are presented as mean ± S.D. B) Transcription initiation complexes encoded by pAC154-dual-dCas9VP160 plasmids, guided by sgRNAs to the HIF1A binding domains in the first intron of the PDL1 gene, evoke PDL1 overexpression. Western blot illustrates PDL1 and Actin protein levels in U251 cells after transfection with plasmids encoding control (scrambled sequence) sgRNA or HIF1A sgRNAs. Note that transcription initiation complexes guided by sgRNAs to the HIF1A binding domains (A), (B), and (A) with (B) simultaneously increased PDL1 expression by 1.5, 3.9 and 1.8 folds, respectively, compared to the control (PDL1 expression in the presence of transcription initiation complexes guided by scrambled sgRNA). In each experiment, PDL1 expression was normalized to the Actin expression.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control, Binding Assay, Over Expression, Transfection, Sequencing, Expressing

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 3: MLN4924 treatment induces up-regulation of HIF1A and PDL1 in glioma cell lines. A) The inhibitory dose- response curves for MLN4924 in established, PDGx and PDGX-stem glioma cell lines. The IC50s are 0.3 ± 0.2 uM (n=4), 2.7 ± 1 uM (n=6), 3 ± 2 uM (n=3), 3 ± 1 uM (n=4), 2.9 ± 0.5 uM (n=4), 0.8 ± 0.2 uM (n =4), 0.2 ± 0.1uM (n=4) for LN221, U251, U87, XD456, JX10, XD456-stem, X14P-stem cell lines, respectively, after treatment with MLN4924 for 5 days. B) Western blots illustrate HIF1A and PDL1 protein levels in nuclear and cytoplasmic fractions in the control and after treatment with MLN4924 (1 uM, 5 days). LaminA/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. C) The graph illustrates normalized PDL1/18S mRNA ratios after treatment with MLN4924 (1uM, 5 days) for different cell lines. Note the significant enhancement of the PDL1/18 mRNA ratio for all cell lines after MLN4924 treatment: 8 ± 3, 25 ± 5, 5 ± 1, 8 ± 3, 4.5 ± fold increase compared to the corresponding control values for U251, Ln229, U87, XD456, JX6 cell lines, respectively, P<0.05, n=3.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 4: Glioma cells treated with MLN4924 decrease T-cell proliferation via utilization of PD1/PDL1 signaling pathway. A) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with U251 cells (1); with U251 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with U251 cells treated with MLN4924 (1uM, for 4 days) (3); with U251 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in the media with/and without an inhibitor of PD1/PDL1 interaction (4 uM). Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (55 ± 8% (n=4) versus 30 ± 8% (n=4), respectively, the difference is significant with P=0.0005). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.0009, n=4). B) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with XD456 cells (1); with UXD456 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with XD456 cells treated with MLN4924 (1uM, for 4 days) (3); with XD456 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in media with/and without an inhibitor of PD1/PDL1 interaction. Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (74 ± 8% (n=4) versus 41 ± 7% (n=4), respectively, the difference is significant with P=0.0003). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.007, n=4).

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Inhibition

Journal: Cancer Research

Article Title: The BH3 Mimetic ABT-737 Induces Cancer Cell Senescence

doi: 10.1158/0008-5472.can-10-1977

Figure Lengend Snippet: Figure 1. Gene transcription changes after ABT-737 treatment. A, microarray analysis of gene expression induced by ABT-737. PV-10 cells were treated in triplicate with DMSO or 10 mmol/L ABT-737 for 24 hours and gene microarray changes documented. B, gene changes associated with senescence. C, qT-PCR analysis of IL-6 and IL-8 transcripts. PV-10 and 22Rv1 cells were treated in triplicate with DMSO, ABT-737 (10 mmol/L) or enantiomer (En., 10 mmol/ L) for 24 hours (mean SD, n ¼ 3). D, the secretion of IL-6 and IL-8 in PV-10 cells treated with DMSO, ABT-737 or enantiomer for 24 hours was determined by ELISA (n ¼ 6, mean SD).

Article Snippet: Conditioned medium was collected following treatment and used at a 2:1 dilution in the human interleukin-6 (IL-6) or IL-8 ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Microarray, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Evaluating the transcriptional regulators of arterial gene expression via a catalogue of characterized arterial enhancers

doi: 10.7554/eLife.102440

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , ChIP DNA Clean & Concentrator kit , Zymo Research , D5205 , .

Techniques: Recombinant, Control, Multiplex Assay, TA Cloning, Software

Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

Journal: Cell Death & Disease

Article Title: The oncogene Etv5 promotes MET in somatic reprogramming and orchestrates epiblast/primitive endoderm specification during mESCs differentiation

doi: 10.1038/s41419-018-0335-1

Figure Lengend Snippet: a Comparison of Etv5 expression levels between mESC lines and somatic cell lines. The relative expression was based on the microarray data from BioGPS database. b The interactions between pluripotency relevant regulators and Etv5 . ChIP-seq and ChIP-chip data with Etv5 as target were extracted from ESCAPE database and used for drawing these interactions. c Growth curve of J1 mESCs stably infected with shCtrl and Etv5 shRNA (shEtv5-7). d RT-qPCR analysis of Etv5 and Tet2 in mESCs stably infected with shCtrl, Etv5 shRNA (shEtv5-7), and shEtv5-7 plus lentiviral Etv5 . Data are shown as mean ± SD ( n = 3). * P < 0.05, *** P < 0.001. Two-way ANOVA with Sidak’s multiple comparisons test was used for c . One-way ANOVA with Dunnett’s multiple comparisons test for d . e Western blotting of TET2 in mESCs stably infected with shCtrl, shEtv5-7, and shEtv5-7 + Etv5 . GAPDH was used as internal control. The relative quantification is also shown. f Dot blot of global 5hmC in mESCs stably infected with shCtrl, shEtv5-7, and shEtv5-7 plus lentiviral Etv5 . The blotting result of serially diluted genomic DNA (100-3.125 ng) was shown (left panel). The same membrane stained with methylene blue as DNA loading control was also presented (right panel)

Article Snippet: For transgenes integration detection, genomic DNA of mouse iPSCs was extracted using TIANamp Genomic DNA Kit (TIANGEN).

Techniques: Comparison, Expressing, Microarray, ChIP-sequencing, ChIP-chip, Stable Transfection, Infection, shRNA, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Dot Blot, Membrane, Staining