Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 3. RASSF10 modulated cell cycle. (a) Cell-cycle distribution was analyzed by FACS flow cytometry in QGY7703 cells and HepG2 cells stably transfected with pcDNA3.1-RASSF10 or pcDNA3.1 vector. Restoration of RASSF10 induced the accumulation of HCC cells in G1 cell cycle phase. The asterisk indicates statistical significance (*Po0.05). (b) Western blot shows the expression of major mediators in cell cycle process including p27, CyclinD1, CDK2 and CDK4.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: Cytometry, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 4. RASSF10 retarded tumor growth in vivo. (a) Subcutaneous tumor growth curve of RASSF10-expressing QGY7703 and HepG2 cells in nude mice was compared with vector (pcDNA3.1) transfected cells. The RASSF10 group showed a retarded tumor growth compared with the vector group (HepG2, P = 0.012; OGY7703, Po0.01). The data are means ± s.d. (n = 8/group). (b) A representative picture of tumor growth in nude mice subcutaneously inoculated with RASSF10 or vector (n = 8/group). (c) Histogram represents mean of the tumor weight from the RASSF10 and vector groups. The asterisk indicates statistical significance (*Po0.05, **Po0.01). (d) Cell cycle mediators including p27, Cycling D1, CDK2 and CDK4 were evaluated in the xenograft tumors by RT-PCR.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation

doi: 10.1038/s41467-024-45308-w

Figure Lengend Snippet: a Motif analysis of ANKRD1 ChIP-seq, assessed and quantified using MEME and DREME software ( https://meme-suite.org/meme/tools/dreme ). Values are expressed as log10 E-value. Shown are the top three transcription factor families enriched in ANKRD1 peak profile. b Prediction of transcription factors binding using the GIGGLE score. GIGGLE represents the similarity between user-defined peak profile with deposited ChIP-seq profiles in the Cistrome DB toolkit ( http://dbtoolkit.cistrome.org/ ). GIGGLE scores for top-ranking AP1 family members on ANKRD1-bound CAF genes are represented with box plots, showing the interquartile range, and the center line representing the median value; the minimum and maximum values delineate the range of data points. Each point represents a separate ChIP-seq dataset. c Predicted 3D structure of ANKRD1-AP1(JUN/FOS)-DNA complex. ANKRD1 3D structure was predicted using Alphafold ( https://alphafold.ebi.ac.uk/ ), the partial crystal structure of JUN/FOS/DNA complex was available at PDB protein databank ( https://www.rcsb.org/structure/1FOS . HADDOCK software ( https://wenmr.science.uu.nl/haddock2.4/ ) was used to dock the two structures. Shown are the clusters with the lowest HADDOCK score. d Van der Waals energy and Electrostatics energy scores for the top eight protein clusters derived from HADDOCK docking of the ANKRD1-AP1(JUN/FOS)-DNA complex. Represented with box plots, showing the interquartile range, and the center line representing the median value; the minimum and maximum values delineate the range of data points. e Schematic view of ANKRD1-JUN predicted interacting residues. The predicted ANKRD1-AP1(JUN/FOS)-DNA complex was used in 3DBionote ( https://3dbionotes.cnb.csic.es/ws ) for predicting the interacting residues between ANKRD1 and JUN protein. ANKRD1 is predicted to interact with JUN through the DNA-binding domain (DBD) and Leucine zipper domain (bZip) of JUN. f Glutathione-conjugated beads were used to immunoprecipitate GST-tagged ANKRD1 (100 ng) recombinant protein mixed with the following recombinant proteins, DNA or AP1 inhibitor: Heat-denatured JUN (100 ng), native JUN (100 ng), HIS-tagged FOS (100 ng), DNA oligo enriched with AP1 consensus motif (50 ng), or T-5224 (20 µM). Western blot analysis for ANKRD1, JUN, and FOS. The experiment was repeated once. g In vitro protein interactions. Glutathione-conjugated beads were used to immunoprecipitate GST-tagged ANKRD1 (100 ng) recombinant protein mixed with the following recombinant proteins: HIS-tagged JUN (truncated form 1-241aa, 100 ng), full-length JUN (100 ng), or HIS-tagged FOS (100 ng). Western blot analysis for ANKRD1, JUN, and FOS. All Co-IP proteins were run in the same nitrocellulose membrane. Similarly, all the inputs (1%) were blotted on the same membrane (also for 6f). Experiment was repeated once.

Article Snippet: GST-tagged ANKRD1 (100 ng), HIS-tagged JUN (truncated form 1-241aa, 100 ng), full-length JUN (100 ng), full-length FOS (100 ng), AP-1 DNA oligo (100 ng) (sc-2501, Santa Cruz), or T-5224 (20uM) (MedChemExpress) were mixed with Glutathione-conjugated beads in binding buffer (150 mM NaCl, 100 mM Tris ph8, 0.5% NP40, 10% Glycerol) for overnight (ON) at 4 °C on a rotating platform.

Techniques: ChIP-sequencing, Software, Binding Assay, Derivative Assay, Recombinant, Western Blot, In Vitro, Co-Immunoprecipitation Assay, Membrane

Journal: Oncogene

Article Title: 15-Lipoxygenase-2 gene regulation by its product 15-(S)-hydroxyeicosatetraenoic acid through a negative feedback mechanism that involves peroxisome proliferator-activated receptor gamma.

doi: 10.1038/sj.onc.1209617

Figure Lengend Snippet: Figure 5 Electrophoretic mobility shift analysis of peroxisome proliferator-activated receptor g (PPARg) and RORa1 protein binding to the 32P-labeled-15 lipoxygenase-2 (15-LOX-2) oligonu- cleotide (560 to 596 bp) in Prostate epithelial cells (PrEC) and Prostate carcinoma cells (PC-3). The protein-DNA complexes that were supershifted (arrows) by the addition of an antibody that recognizes PPARg. (a) Lanes 1 and 2, PrEC nuclear extract without and with dnPPARg; lanes 3 and 4, PC-3 nuclear extract without and with dnPPARg; lane 5, PC-3 nuclear extract with the consensus PPARg probe; lane 6, PC-3 nuclear extract incubated with the mutant PPARg probe. Consensus PPRE or mutant oligonucleotides were used as unlabeled competitors to determine the specificity of the binding reaction in PC-3. (b) Lane 1, 32P- labeled-RORE consensus site free probe; lanes 2, 3 and 4, PC-3 nuclear extract with increasing concentrations of unlabelled RORE oligonucleotide as competitor; lane 5, 32P-labeled-15-LOX-2 free probe; lanes, 6, 7 and 8, PC-3 nuclear extract in the presence of increasing concentrations of unlabeled 15-LOX-2 oligonucleotide as competitor; lanes, 9 and 10, PC-3 nuclear extract with 15-LOX-2 probe and without and with the RORa antibody; lane 11, PC-3 nuclear extract with 15-LOX-2 probe and RORa antibody in presence of 50 unlabeled 15-LOX-2 oligonucleotide.

Article Snippet: Wild-type PPARg and mutant gel shift oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Electrophoretic Mobility Shift Assay, Protein Binding, Labeling, Incubation, Mutagenesis, Binding Assay