Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments modulate Notch receptors expression and activity. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (A) TALL-1 or (C) MOLT3 cells treated for 48 h with 2 μM GSKJ4 or with DMSO. (B) Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panel) and HA and β-actin protein levels (lower panel) in TALL-1 cells transfected with HA-tagged EZH2 expression vector (HA-EZH2) or with the empty control vector. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (D) TALL-1 or (E) MOLT3 cells treated for 48 h with 5 μM A-485 or DMSO. Data represent mean values of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, ** P < 0.01, *** P < 0.001. Uncropped western blots related to this figure are displayed in .

Article Snippet: The expression vector PIRVNeoSV containing the human c-Myc cDNA coding sequence (c-Myc) was kindly provided by Dr. Giuseppe Giannini (Sapienza University, Rome, Italy). pCMV3-HA vector containing the human EZH2 coding sequence (HA-EZH2) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: Genes & genetic systems

Article Title: Scarce adenylation in bacteriophage T4 mRNAs.

doi: 10.1266/ggs.77.219

Figure Lengend Snippet: Fig. 3. The length distribution of the 3’-5’ junction of (A) circular soc RNA or (B) ligated uvsY RNA-16S rRNA. After reverse tran- scription of total RNA from T4-infected cells, cDNA was utilized for amplification by PCR as described in Materials and Methods. After amplified DNAs were electrophoresed through a 5% polyacrylamide gel, DNA fragments in a size range of 210– 280 bp for soc and of 250–320 bp for uvsY were eluted from gel, heat-denatured and utilized for the length analysis. The length analysis was performed as follows. The csoc-up primer for soc or 16S5’ primer for uvsY was labeled at its 5’-end with 32P using T4 polynucle- otide kinase, annealed to heat-denatured DNA and subjected to chain-elongation by Taq DNA polymerase at 72°C for 160 sec. The chain-elongation reaction was carried out in a mixture contained 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, 250 µg/ml bovine serum albu- min, 2% sucrose and 0.2 mM each of four deoxyribonucleotides. The products were heat-denatured and analyzed through a 5%-poly- acrylamide gel containing 6 M urea (lanes 1 and 3). Preparation of reference strands was as follows: pCSOC#5 or pY-EC16#4 DNA was used as a template for PCR with a respective set of primers (csoc-dw and csoc-up for soc, and uvsY3’ and 16S5’ for uvsY). The amplified fragments were heat-denatured and used as a template for strand elongation in the same manner as above. The reference strands were run in parallel (lanes 2 and 4). Sequence ladders obtained by dideoxynucleotide method (Innis et al., 1988) with either a combination of pCSOC#5 and csoc-up primer or a combination of pY-EC16#4 and 16S5’ primer were also run in each left four lanes (G, A, T, C) of figure A or B, respectively. In the left margins, sequences at the border of cloned DNA and vector are shown.

Article Snippet: The cDNAs were amplified by PCR containing Thermococcus kodakaraensis DNA polymerase (KOD dash, Toyobo co. ltd.) with the same primer as used for cDNA synthesis and an upstream primer.

Techniques: Infection, Amplification, Labeling, Acrylamide Gel Assay, Sequencing, Clone Assay, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: rs4903064 resides in a renal cancer–associated regulatory element. A , ATAC-seq tracks from primary renal cancer cells at the DPF3 locus. The top track indicates SNPs in high LD with rs4903064 (r 2 > 0.8). ATAC-seq was performed in cells from three individuals with different rs4903064 genotypes (TT, CT, CC). KIRC RE indicates r egulatory e lements defined by ATAC-seq in ccRCC TCGA samples. B , zoomed in view of ( A ). SNP rs4903064 resides in open chromatin and in KIRC_67261. Please note that open chromatin is only detected in cells carrying the risk allele C. C , sequences of the two alleles at rs4903064. The risk allele C creates a putative hypoxia-responsive element (HRE). D , allele-specific qPCR assay for rs4903064 indicates a shift toward the risk allele C in DNA fragments captured by ATAC experiments in tumor cells ( red triangle ) or tubular cells exposed to 1 mM DMOG ( orange triangle ) when compared to input DNA or DNA from ATAC experiments in untreated tubular cells. Cells were isolated from an individual heterozygous for rs4903064. E , allelic ratio of rs4903064 in ATAC samples from tubular cells isolated from three individuals heterozygous for the SNP performed in triplicates. Cells were exposed to 1 mM DMOG for 16 h or left untreated. Bars indicate mean values ± SD. t test, ∗∗∗∗ p < 0.0001. F , allelic ratio of rs4903064 in FAIRE samples from cells of four heterozygous individuals. Cells were exposed to 1 mM DMOG for 16 h, and values were normalized to corresponding input DNA. The mean value for each individual is shown. Bars indicate mean values ± SD. ∗one sample t test, p < 0.05. G , reporter assay in HeLa cells using the pGL3 promoter vector with or without a 415 bp sequence covering the protective or the risk allele of rs4903064. Cells were treated with 1 mM DMOG for 16 h as indicated. Bars indicate mean values ± SD of one experiment performed in triplicates. H , HeLa cells were depleted for the indicated HIF-α isoforms using siRNA and transfected with the reporter plasmid including the risk allele. Cells were exposed to 1 mM DMOG for 16 h. Reporter activity was normalized to the activity of co-transfected β-galactosidase. Bars indicate mean values ± SD of one experiment performed in triplicates. I and J , DPF3 protein levels are high in HIF-positive ccRCC tumors. A total of 330 ccRCC tumors were stratified according to the immunoreactive score for HIF-1α, HIF-2α, and DPF3 staining in no, low, medium, or strong staining. Fractions of DPF3 signals are shown for each subgroup of HIF staining. Pearson χ 2 -test; p = 1.8 × 10 −8 for HIF-1α ( I ) and p = 0.003 for HIF-2α ( J ). ATAC, Assay for Transposase-Accessible Chromatin; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; FAIRE, formaldehyde-assisted isolation of regulatory elements; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Isolation, Reporter Assay, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: HIF binding to the RCC risk allele. A , HIF ChIP qPCR results for the DPF3 locus. Experiments were performed from cells isolated from 11 TT, 11 CT, and 1 CC individuals. Bars indicate mean values ± SD. Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01. B , binding of HIF to the control locus at EGLN3 is not affected by the rs4903064 genotype. Bars indicate mean values ± SD. C , HIF ChIP-seq tracks in two different ccRCC cell lines at the rs4903064-associated enhancer (TT: RCC4 and CC: individual 40,911,432 from Yao et al. ). HIF binding is only detectable in cells from a patient carrying the C allele ( red , HIF-1α: 12 × C from 12 reads in total, HIF-2α 99 × C from 99 reads in total). D , same tracks as in ( C ) covering the EGLN3 control locus. E and F , H3K27ac ChIP-seq tracks from corresponding normal ( blue ) and ccRCC ( red ) tissue indicates increased activity of the disease-associated regulatory element at DPF3 ( E , highlighted in yellow ) and of the EGLN3 locus ( F ) in tumors. G , H3K27ac ChIP-seq tracks for two different ccRCC cell lines at the rs4903064-associated enhancer (TT: 786-O and CC: individual 40,911,432 from Yao et al. ). Levels of the active enhancer mark H3K27ac are high in the individual carrying the C allele and reduced upon VHL re-expression. H , same tracks as in ( G ) covering the EGLN3 control locus. Note that H3K27ac levels are reduced upon VHL re-expression in both cell lines. ccRCC, clear cell renal cell carcinoma; ChIP, chromatin immunoprecipitation; DPF3, double PHD fingers 3; EGLN3, Egl-9 family hypoxia inducible factor 3; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism; VHL, von Hippel-Lindau.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Binding Assay, ChIP-qPCR, Isolation, Control, ChIP-sequencing, Activity Assay, Expressing, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Allele specific interactions at rs4903064. A – C , allele-specific qPCR on ChIP samples generated from primary tubular cells exposed to 1 mM DMOG for 16 h. A , HIF ChIP indicates enrichment for the C allele in DNA fragments captured with HIF-1α or HIF-1β antibodies. n = 7 individuals. B , H3k27ac as a marker for active chromatin is enriched at the C allele. n = 4 individuals. C , RNApol2 is enriched at the C allele of rs4903064 indicating increased interaction with the transcriptional machinery from this allele. n = 4 individuals. D , quantification of the results in ( A – C ). The allelic ratio is shifted to the risk allele C, which indicated increased interaction with HIF and the transcriptional machinery as well as increased activity of this allele. Values are mean ± SD. One sample t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ChIP, chromatin immunoprecipitation; DMOG, dimethyloxalylglycine; HIF, hypoxia-inducible transcription factor.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Generated, Marker, Activity Assay, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Regulation of DPF3 expression and effect of DPF3 on tubular cell growth. A , relative expression levels of DPF3 in primary renal tubular cells exposed to 1 mM of DMOG for 16 h. Samples were stratified according to the rs4903064 genotype (TT = 42, CT/CC = 44). Values were normalized to expression levels of the housekeeping gene HPRT and to values from corresponding untreated cells. Each dot represents the mean value from cells of one individual. qPCR was performed in duplicates per individual. Bars indicate mean values ± SD. Student’s t test, ∗∗∗∗ p < 0.0001. B , DPF3 expression levels in isolated tumor cells compared to corresponding untreated tubular cells from 8 (TT) and 14 (CT/CC) individuals, respectively. Bars indicate mean values ± SD. One sample t test, ∗ p < 0.05. C , top , allelic ratios of rs4903064 in cells from heterozygous individuals exposed to DMOG 1 mM or left untreated. One sample t test, ∗∗∗ p < 0.001. Bottom , allelic ratios of rs4903064 in ccRCC cells with high (CA9+) or low (CA9−) CA9 expression as a marker for HIF-1α activity. Values are mean from technical triplicates, and each dot represents values derived from one individual. Bars indicate mean values ± SD. t test, ∗ p < 0.05. D , Western blot for DPF3 and beta actin from lysates of different clones of HKC-8 cells with or without defective DPF3. E , Western blot for DPF3 and beta actin from lysates of two different huPTC lines transfected either with nontargeting (nt) guides or guides targeting DPF3. F , proliferation assays of the clones of cells indicated in ( D ). Dark lines and dots indicate the mean value from the three different clones of cells, which were tested in biological triplicates. Light gray and light blue lines indicate individual values for the three different clones of cells. G , proliferation assay from cells indicated in ( E ). H , Western blot for DPF3 and beta actin from lysates of primary renal tubular cells (PTC) transfected either with nontargeting (nt) guides or guides targeting DPF3. Cells were harvested at day 3 after transfection. I , cyst volume of PTC 7 days after plating in a Matrigel matrix (n = 3 wells per condition, 4 quadrants per well). J , representative images of the cysts in the Matrigel matrix. Values are mean ± SD. t test, ∗ p < 0.05, ∗∗ p < 0.01 for F , G , and I . CA9, carbonic anhydrase 9; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; huPTC, human urinary primary tubular cells.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Expressing, Isolation, Marker, Activity Assay, Derivative Assay, Western Blot, Clone Assay, Transfection, Proliferation Assay

Journal: BMC Cell Biology

Article Title: Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

doi: 10.1186/1471-2121-11-44

Figure Lengend Snippet: ITGA5 Controls IGF2/IGFBP2 Expression in hMSCs . Adult hMSCs were transduced with shITGA5, shITGB1 or a non relevant shRNA (shNR), total RNA was used for quantitative RT-PCR analysis of IGF2 ( A ) and IGFBP2 ( B ). Adult hMSCs were transduced with a lentiviral vector encoding ITGA5, or treated with the agonist peptide CRRETAWAC (100 μg/ml) (CRRETAWAC) or with a non relevant control peptide (GRGESP; 100 μg/ml), or with a conformation-dependent anti-α5 monoclonal antibody (SNAKA51; 10 μg/ml), and IGF2 and IGFBP2 mRNA expression was determined by quantitative RT-PCR analysis ( C-D ). Results are expressed as mean ± SD of treated over control ratio after normalization to 18 S expression. *: significant difference with untreated cells ( P <0.05).

Article Snippet: Non relevant shRNA (scrambled sequence that does not lead to specific degradation of any known cellular mRNA), ITGB1 shRNA and human FAK shRNA lentiviral particles (mixtures of viral particles containing 3 target-specific constructs that encode shRNA designed to knock down gene expression) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Transduction, shRNA, Quantitative RT-PCR, Plasmid Preparation, Control

Journal: BMC Cell Biology

Article Title: Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

doi: 10.1186/1471-2121-11-44

Figure Lengend Snippet: Inhibition of ITGA5-Induced Signalling Abrogates IGF2/IGFBP2 Expression in hMSCs . Adult hMSCs transduced with a lentiviral vector encoding ITGA5 or treated with the agonist peptide (CRRETAWAC; 100 μg/ml), and control cells were transiently transfected with a specific shRNA targeting FAK. Total RNA was collected and used for quantitative RT-PCR analysis of IGF2 ( A ) and IGFBP2 ( B ). Adult hMSCs were treated with the MEK inhibitor U0126 (10 μM), or the PI3K inhibitor wortmannin (50 nM) for 24 hours and IGF2 and IGFBP2 mRNA levels were determined by quantitative RT-PCR analysis ( C-D ). Results are expressed as mean ± SD of treated over control ratio after normalization to 18 S expression. *: significant difference with untreated cells ( P <0.05).

Article Snippet: Non relevant shRNA (scrambled sequence that does not lead to specific degradation of any known cellular mRNA), ITGB1 shRNA and human FAK shRNA lentiviral particles (mixtures of viral particles containing 3 target-specific constructs that encode shRNA designed to knock down gene expression) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Inhibition, Expressing, Transduction, Plasmid Preparation, Control, Transfection, shRNA, Quantitative RT-PCR