Journal: iScience

Article Title: Targeting HOTAIRM1 ameliorates glioblastoma by disrupting mitochondrial oxidative phosphorylation and serine metabolism

doi: 10.1016/j.isci.2022.104823

Figure Lengend Snippet:

Article Snippet: NRAS (F155) Mouse monoclonal antibody , Santa Cruz Biotechnology , # sc-31; RRID: AB_628041.

Techniques: Labeling, Control, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, cDNA Synthesis, Isolation, Reporter Assay, Extraction, Knockdown, Microarray, shRNA, Negative Control, Software, Membrane, Sterility

Journal: Cancer Cell

Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

doi: 10.1016/j.ccell.2017.08.010

Figure Lengend Snippet: Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p < 0.001. Irrelevant bands were omitted from gels (B). Areas in which lanes were omitted are indicated by white space between lanes. See also Figure S7 .

Article Snippet: Primary antibodies against γH2AX, p-p53, GAPDH, PCNA, pATM, pCHK1, pJNK1/2, pCHK2, pATR, pBRCA1, cleaved-Casp1, cleaved-Casp3, cleaved-Casp8, RIPK1, RIPK3, total-JNK1/2, p-p38, p38, cIAP1, cIAP2, XIAP (all Cell Signaling Technology), total-Casp8, p-cJUN, pAKT1/2/3, total AKT (SantaCruz), total-ATM (Sigma), total-cJUN (BD Bioscience), HOIP (R&D), SHARPIN (Proteintech), HOIL-1 (Walczak lab) and Casp8 C15 (provided by Dr. P. Krammer, Heidelberg), were incubated at 4°C overnight under shaking conditions.

Techniques: Staining, Mutagenesis, Labeling, Immunoprecipitation, Western Blot, Activation Assay, Fractionation

Journal: Cancer Cell

Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

doi: 10.1016/j.ccell.2017.08.010

Figure Lengend Snippet:

Article Snippet: Primary antibodies against γH2AX, p-p53, GAPDH, PCNA, pATM, pCHK1, pJNK1/2, pCHK2, pATR, pBRCA1, cleaved-Casp1, cleaved-Casp3, cleaved-Casp8, RIPK1, RIPK3, total-JNK1/2, p-p38, p38, cIAP1, cIAP2, XIAP (all Cell Signaling Technology), total-Casp8, p-cJUN, pAKT1/2/3, total AKT (SantaCruz), total-ATM (Sigma), total-cJUN (BD Bioscience), HOIP (R&D), SHARPIN (Proteintech), HOIL-1 (Walczak lab) and Casp8 C15 (provided by Dr. P. Krammer, Heidelberg), were incubated at 4°C overnight under shaking conditions.

Techniques: Purification, Imaging, Mutagenesis, Recombinant, Staining, SYBR Green Assay, Microarray, RNA Expression, RNA Sequencing Assay, Methylation, Labeling, Software, Light Microscopy

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: Accurate Molecular Classification of Renal Tumors Using MicroRNA Expression

doi: 10.2353/jmoldx.2010.090187

Figure Lengend Snippet: Classification of kidney tumors using expression levels of six microRNAs (microarray data). A: Classification proceeds in two steps, following the cluster structure of the histological types (Figure 2). First, samples are classified into either the oncocytoma/chromophobe pair, or the conventional/papillary pair, using expression levels of hsa-miR-210 and hsa-miR-221 (B). In the second step, oncocytoma is differentiated from chromophobe using expression levels of hsa-miR-200c and hsa-miR-139-5p (C), and conventional is differentiated from papillary using expression levels of hsa-miR-31 and hsa-miR-126 (D). Independent test samples included oncocytoma samples (n = 19, red stars), chromophobe tumors (n = 14, black/yellow diamonds), conventional tumors (n = 17, blue squares), and papillary tumors (n = 6, green circles). The gray shaded regions indicate the thresholds for classification for each pair of microRNAs, indicating in each case the right branch in the binary classification tree (A). The 71 samples that were used for training the thresholds (see Methods and Figure 3) are shown in faded symbols in the background.

Article Snippet: 31 , 37 Briefly on Slide E coated microarray slides (Schott Nexterion, Mainz, Germany) >900 DNA oligonucleotide probes representing microRNAs were spotted in triplicate using the BioRobotics MicroGrid II microarrater (Genomic Solutions, Ann Arbor, MI) according to the manufacturer's directions.

Techniques: Expressing, Microarray

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: Accurate Molecular Classification of Renal Tumors Using MicroRNA Expression

doi: 10.2353/jmoldx.2010.090187

Figure Lengend Snippet: Validation by qRT-PCR. The qRT-PCR validation set included 32 tumor samples: 8 oncocytoma tumors (red stars), 8 chromophobe tumors (yellow diamonds), 8 conventional tumors (blue squares), and 8 papillary tumors (green circles). For each decision point (Figure 4A), the plot on the left side (A, C, or E) shows the log2 expression ratio of the two microRNAs used at this node as measured by microarray (horizontal, calculated as the log2 ratio of the normalized fluorescence signal) and by qRT-PCR (vertical, calculated as the difference in normalized Ct values). In each plot, the vertical line demarcates the classification threshold trained on the microarray data, while the horizontal line indicates the classification threshold that was chosen for the qRT-PCR data, based on the microarray threshold (Methods). The plots on the right side (B, D, or F) show the qRT-PCR data (normalized Cts) of the two microRNAs in the samples of the relevant subtypes at each node. The gray shaded regions indicate the thresholds for classification for each pair of microRNAs, indicating in each case the right branch in the binary classification tree (Figure 4A). A: Log2 expression ratio of [hsa-miR-221/hsa-miR-210] in microarray and qRT-PCR. Correlation coefficient was 0.92. B: Normalized Cts of hsa-miR-221 and hsa-miR-210 in 32 samples. C: Log2 expression ratio of [hsa-miR-139-5p/hsa-miR-200c] in microarray and qRT-PCR. Correlation coefficient was 0.86. D: Normalized Cts of hsa-miR-139-5p and hsa-miR-200c in 16 oncocytoma and chromophobe samples. E: Log2 expression ratio of [hsa-miR-126/hsa-miR-31] in microarray and qRT-PCR. Correlation coefficient was 0.98. F: Normalized Cts of hsa-miR-126 and hsa-miR-31 in 16 conventional and papillary samples.

Article Snippet: 31 , 37 Briefly on Slide E coated microarray slides (Schott Nexterion, Mainz, Germany) >900 DNA oligonucleotide probes representing microRNAs were spotted in triplicate using the BioRobotics MicroGrid II microarrater (Genomic Solutions, Ann Arbor, MI) according to the manufacturer's directions.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Microarray, Fluorescence

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: Accurate Molecular Classification of Renal Tumors Using MicroRNA Expression

doi: 10.2353/jmoldx.2010.090187

Figure Lengend Snippet: Classification of Renal Tumors Using Expression Levels of 6 MicroRNAs

Article Snippet: 31 , 37 Briefly on Slide E coated microarray slides (Schott Nexterion, Mainz, Germany) >900 DNA oligonucleotide probes representing microRNAs were spotted in triplicate using the BioRobotics MicroGrid II microarrater (Genomic Solutions, Ann Arbor, MI) according to the manufacturer's directions.

Techniques: Expressing, Biomarker Discovery, Microarray