Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Cell reports

Article Title: Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates

doi: 10.1016/j.celrep.2020.107599

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used at the concentration indicated by manufacturer’s protocol: CaM Kinase II alpha (6G9) (NB100–1983), LMX1A (NBP1–81303) Novusbio; SYNAPSIN (106 001), HOMER (160 003) Synaptic System; EGFR (Ab231), FGFR1 phosphoY654 (Ab59194), TBR1 (Ab31940), REELIN (Ab18570), CYCLIN D1 (Ab10540), FGFR2 (Ab10648), BMPR1A (Ab38560) Abcam; HES1 (11988), p-SMAD1/5 (9516), CYCLIN D1 (2926), pERK1/2 (4370), FGFR1 (9740) Cell Signaling Technology; PAX6 (PRB-278P) BioLegend; NESTIN (MAB1259), OTX2 (AF1979), PDGFR alpha (AF1062; AF307), SOX2 (AF2018; MAB2018), SOX21 (AF3538), TuJ1 (MAB1195), EGFR (AF1280), O4 (MAB 1326) R&D Systems; GFAP (Z 0334) DAKO; HES5 (sc-13859), CUX1 (sc-13024), TLE4 (sc-9125), FGFR3 (sc-9007), LHX2 (sc-19344) Santa Cruz Biotechnology; FOXP2 (ABE73), TBR1 (AB2261), REELIN (MAB5366) Millipore; FOXG1 (M227) Takara/Clontech; anti GAD65/67 was kindly gifted by Dr. Christian Geis, Hans Berger Department of Neurology, Jena University Hospital, Germany ( ).

Techniques: Virus, Plasmid Preparation, Recombinant, Transfection, Antibody Labeling, In Vitro, Microarray, Gene Expression, Derivative Assay, Software

Journal: PLoS ONE

Article Title: A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

doi: 10.1371/journal.pone.0117744

Figure Lengend Snippet: a) microfluidic design: the device consists of two PDMS layers: flow (blue) and control (red). The chip is an array of eight rows by 48 columns for 384 unit cells. Each unit cell is composed of: two antibody chambers divided by a reaction chamber (1–2), 4 MITOMI buttons (3), a valve that segregates the unit cells (4), a valve that separates antibody and reaction chambers (5) and a valve for releasing pressure in the antibody chambers (6). b) The PDMS chip is aligned to an epoxy-functionalized slide onto which primary and secondary antibodies were spotted. c) Assay details: schematic of the unit cell and cross section of a button region: i) functionalization of the surface: BSA-biotin is flowed though the chip followed by neutravidin. Next, the buttons are closed and BSA-biotin flowed again to passivate all neutravidin molecules except for those located underneath the MITOMI buttons, ii) the biotinylated primary antibody is allowed to diffuse into the MITOMI detection chamber and is bound by neutravidin immobilizing it in the MITOMI detection regions, iii) the sample is flown through the device and antigens are captured by the surface immobilized antibodies, iv) finally, the fluorescently labeled secondary antibody is allowed to diffuse into the MITOMI area, binds to the antigen if present, and is trapped by MITOMI. The entire device is then quantitated using a DNA microarray scanner.

Article Snippet: Neutravidin biotin-binding protein and the biotinylated bovine serum albumin BSA were purchased from Thermo Scientific (29130 and 31000 respectively).

Techniques: Control, Labeling, Microarray

Journal: Cell stem cell

Article Title: Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

doi: 10.1016/j.stem.2017.04.009

Figure Lengend Snippet: Figure 7. a-ITGA7 Impairs GBM Tumor Growth and Invasion In Vivo (A) Luciferase activity detected with a Xenogen IVIS 100 small animal in vivo imaging system, 5 days post-sc transplantation of 5 X 105 GSC1-LUC cells in mice either treated with anti-ITGA7 (a-ITGA7) antibody or isotype control (i.p.). (B) Analysis of sc GSC1-LUC tumor growth over time in mice treated either with isotype control or with a-ITGA7 antibody (1.4A12). Shown is the average photon count and SEM of n = 6 mice/group measured by Xenogen IVIS 100small animal in vivo imaging system (***p< 0.001, two-tailed Student’s t test). See also FigureS7. (C) Analyses of the tumor volumes 5 months after ceasing the anti-ITGA7 treatment. Shown is the size of the sc tumors of n = 6 mice/group measured by calliper (**p < 0.01, two-tailed Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PE Mouse monoclonal anti EphA2 (clone 371805) R&D Cat#FAB3035P; RRID:AB_11128496 APC Mouse monoclonal anti CD44 BD Biosciences Cat# 560890 Mouse monoclonal anti CD44 (clone DF1485) DAKO Cat#M7082; RRID:AB_2076596 Mouse monoclonal anti ITGA7 Miltenyi Bioteck Cat#130-103-774 Mouse monoclonal anti murine ITGA7 (clone 6A11) Acris Cat#AM20012AF-N; Please See Table S3 for Reverse Phase Protein Microarray Antibodies used N/A N/A Biological Samples GSC (Glioblastoma Stem Cells) (Marziali et al., 2016; Ricci-Vitiani et al., 2008) N/A Chemicals, Peptides, and Recombinant Proteins EGF Peprotech Cat#100-15 bFGF Peprotech Cat#100-18B Apotransferrin Sigma Cat#T2252 Putrescine Sigma Cat#P5780 Sodium Selenite Sigma Cat#S5261 Bovine Serum Albumin Sigma Cat#A2153 Progesterone Sigma Cat#P8783 Insulin Sigma Cat#I3536 HAT Supplement 50X Thermo Scientific Cat#21060017 HT Supplement 100X Thermo Scientific Cat#11067030 BM Condimed H1 Hybridoma Suppl.

Techniques: In Vivo, Luciferase, Activity Assay, In Vivo Imaging, Transplantation Assay, Control, Two Tailed Test

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Control, Molecular Weight, Marker

Journal: medRxiv

Article Title: Inhibition of SHP2 ameliorates psoriasis by decreasing TLR7 endosome localization

doi: 10.1101/2020.09.28.20202861

Figure Lengend Snippet: (A) Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). (B) Expression of PTPN11 in human PBMCs from psoriatic patients (n=14) and normal controls (n=16). (C) Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls. (D) Representative SHP2 staining in skin sections from psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm. (E) The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). (F) Representative p-ERK staining of skin sections from psoriatic patients and normal controls. Scale bars: 200 μm. (G) Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back from C57BL/6J mice at day 5 (n=6/group). Data were normalized to GAPDH expression. (H) Representative histological sections of IMQ-treated or non-treated dorsal back from C57BL/6J mice at day 5. Scale bar: 100 μm. Data represent mean ± SEM. P values are determined by Two-tailed Mann-Whitney U test (A and B) or Two-tailed Student’s t test (E and G). * P <0.05, ** P <0.01.

Article Snippet: For immunohistochemistry, the human and mouse skin paraffin sections were deparaffinized, rehydrated, and antibody retrieved with sodium citrate, blocked, then stained with anti-SHP2 (Santa Cruz, catalog sc-7384), anti-ERK (Cell Signal Technology, catalog 4695), anti-CD68 (Cell Signal Technology, catalog 76437), anti-p-p65 (Cell Signal Technology, catalog 3033), anti-Ki67 (Abcam, catalog ab15580) were used at 1:100 overnight at 4°C.

Techniques: Expressing, Microarray, Western Blot, Derivative Assay, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: Nanoparticle-enabled cytosolic delivery of 2’3’-cGAMP activates the STING pathway in neuroblastoma cells. (A) Integrated molecular analysis of mRNA expression of genes from the pediatric neuroblastoma TARGET dataset that have been distinguished by functional significance and clustered into evenly split tertiles based on high (upper tertile, n=47), intermediate (median tertile, n=47), and low (bottom tertile, n=47) TMEM173 mRNA expression. (B) TMEM173 mRNA expression in MYCN non-amplified and amplified samples profiled by microarray in the TARGET pediatric neuroblastoma (n=55) datasets. Data were accessed through the cBioPortal. Mann-Whitney U test (two-tailed) was used for statistical comparison.(C) Schematic representation of STING-NPs designed to enhance cytosolic delivery of cGAMP via endosomal escape, resulting in potent activation of STING signaling. (D) Neuroblastoma cell lines (Neuro-2a, 9464D, SK-N-SH, and LAN-1) were treated with vehicle (PBS), empty nanoparticles (NP), 200 nM (or 400 nM for 9464D) cGAMP, or STING-NPs for 48 hours; cells were collected for western blot analysis using anti-IRF3 and anti-phospho-IRF3 (p-IRF3) antibodies. Gel loading was normalized for equal actin; representative blots from one of two independent experiments. The relative density of bands is shown under each immunoblot, after normalization to the levels of actin. (E) qRT-PCR gene expression of IFNB1, TNF, CXCL10, and IL12 in neuroblastoma cell lines at 6, 24 and 48 hours after treatment with cGAMP or STING-NPs. (n=2, data shown as mean±SD; data was analyzed by two-way ANOVA followed by Dunnett’s posthoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate a statistically significant difference relative to vehicle (PBS). ANOVA, analysis of variance; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Expressing, Functional Assay, Amplification, Microarray, MANN-WHITNEY, Two Tailed Test, Comparison, Activation Assay, Western Blot, Quantitative RT-PCR, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: STING activation in neuroblastoma generates an immunogenic, tumoricidal, and T-cell inflamed tumor microenvironment. Mice with 200 mm 3 Neuro-2a tumors were administered PBS or STING-NPs (10 µg cGAMP) intratumorally three times, spaced 3 days apart, and tumors were harvested 48 hours following the last treatment. (A) qRT-PCR analysis of IFNB1, TNF, CXCL10, and IL12 gene expression in injected Neuro-2a tumors. n=5 to 7 mice per group represented as mean±SEM, *p<0.05, **p<0.01 indicate a statistically significant difference using a two-tailed Mann-Whitney U test. (B) Neuro-2a tumor lysates (n=4–5) were analyzed using western blot for phospho-STAT1 (p-STAT1), IRF3, and phospho-IRF3 (p-IRF3), caspase 3, and cleaved caspase 3. Gel loading was normalized for equal actin. The density of bands is shown under each immunoblot, after normalization to the levels of actin. (C/D) Immunohistochemical staining of tumor sections for cleaved caspase-3 (apoptosis), Ki-67 (proliferation), CD8, and FoxP3 and corresponding quantification of staining intensity using ImageJ software. Data shown as mean±SD for n=3 Neuro-2a tumors, ***p<0.001, ****p<0.0001 indicate a statistically significant difference using a Student’s t-test. cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Quantitative RT-PCR, Gene Expression, Injection, Two Tailed Test, MANN-WHITNEY, Western Blot, Immunohistochemical staining, Staining, Software, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Expression of CDK11 and CK2 protein complex members in untransformed and malignant breast cells. (A) Immunoblot analysis of cultured breast cell lines, as indicated above the blots. Proteins detected are indicated on the right side of the blots. Actin signal was used as the loading control. (B) Indirect immunofluorescent detection of CDK11, CK2α, and CK2α′ (red color) in breast cell lines. Cell lines are indicated above each set of images and proteins detected are indicated on the left side of the images. Blue, 4′,6-diamidino-2-phenylindole-stained nuclei. Scale bar: 100 μm. (C) Immunohistochemical detection of CDK11 proteins in human normal and malignant breast tissue. Type of breast tissue indicated on the left side of the images. Magnification indicated above the images; dotted ellipse, portion of the 100× image that is shown at 400×. Scale bars: 400 μm for 100× and 100 μm for 400× images. (D) Human microarray tissues stained for CDK11 were scored by two independent observers. The average value was taken and the results plotted for normal ( n = 16) versus triple-negative breast cancer (TNBC; n = 44) tissues. Box, first to third (Q1 to Q3) quartiles; diamond, mean; line inside box, median; whiskers, minimum and maximums of data range. CDK, cyclin-dependent kinase; CK2, casein kinase 2.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Cell Culture, Control, Staining, Immunohistochemical staining, Microarray

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: mRNA expression levels in nontransformed and malignant breast cells a

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: RNA expression levels in normal breast and breast cancer subtypes. Normalized RNAseq read count data for PAM50 breast cancer subtypes and normal breast from The Cancer Genome Atlas were analyzed for CDK11 and CK2 protein complex genes as shown above each plot. Box, first to third (Q1 to Q3) quartiles; line inside box, median; whiskers, 1.5 maximum interquartile range. Normal, n = 95; basal, n = 141; Her2, n = 67; LumA, n = 421; LumB, n = 192. CDK, cyclin-dependent kinase; CK2, casein kinase 2; Her2, human epidermal growth factor receptor 2; LumA, luminal A; LumB, luminal B.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: RNA Expression

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Immunoblot analyses following small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells. Immunoblot analysis of MDA-MB-231 and SUM-149 cell lysates following small interfering RNA (siRNA) transfection. Transfected siRNAs are indicated above the blots, proteins detected are indicated on the right side of the blots. CDK11 p110 , cyclin L1α, cyclin L2α, and CK2αα′β lysates are 72 hours post transfection; caspase 3, Bcl-xL, and survivin lysates are 96 hours post transfection. Actin signal was used as the loading control. CDK, cyclin-dependent kinase; CK2, casein kinase 2.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Small Interfering RNA, Transfection, Control

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Protein expression levels following small interfering RNA transfection

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Small Interfering RNA

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: mRNA expression levels in small interfering RNA transfected cells

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Small Interfering RNA, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells decreases cell viability and inhibits clonal survival. (A) Breast cancer cells were transfected with 30 nM single small interfering RNA (siRNA) or 15 nM each of the two siRNAs combined as indicated. After 96 hours, cell viability was determined relative to the untreated cells. Means ± standard errors (SEs) are presented. * P <0.05. ** P <0.01, *** P <0.001 relative to untreated. ^ P = 0.055, # P <0.05, ## P <0.01, ### P <0.001 relative to siCtrl. (B) Triple-negative breast cancer (TNBC) cells were transfected twice with 30 nM single siRNAs or 15 nM each of the two siRNAs combined as indicated and as described in . Seven days after the second transfection, cell colonies were stained and counted. Means ± SE are presented. $ P <0.0001 relative to siCtrl and untreated. (C) Representative crystal violet stained colonies on 35 mm plates 7 days after the second siRNA transfection as described in (B). Cell lines are indicated above the plate images and siRNA transfections are indicated to the left of the plate images. CDK, cyclin-dependent kinase; CK2, casein kinase 2; si, small interfering.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Small Interfering RNA, Transfection, Staining

Journal: Nature communications

Article Title: Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

doi: 10.1038/ncomms4361

Figure Lengend Snippet: ( a ) MCF-10A cells were infected by the indicated lentiviral particles targeting the indicated genes. Control cells and knockdown cells were subjected to microarray analysis. The presence of the HRD gene signature was analyzed by supervised clustering analysis. ( b ) Modified HR repair assay was performed in MCF-10A cells by transfecting cells with DRGFP DSB substrate plasmid and I-SceI plasmid through electroporation, followed by analysis of GFP-positive cells by flow cytometry 48 to 72 hours later. Student’s t -test was performed from results of three independent experiments as mean + SD. ( c ) The rate of cell survival in response to olaparib was determined by colony formation assay. Each value was relative to control cells without treatment and represents the mean ± SD from three independent experiments. Student’s t -test showed that treatment response differed between BRCA1-PTEN double knockdown cells and single knockdown cells (P<0.001). ( d ) Heat map of the HRD gene signature with the 26 genes most significantly changed in BRCA1-PTEN double knockdown cells compared to single-knockdown-cells. ( e ) Microarray data from 295 breast cancers were clustered into basal-like, Her2-positive (Her2), luminal A, luminal B, and normal breast-like. Gene expression levels of TTK among the different breast cancer subtypes are indicated. ( f ) Quantitative analysis of HR repair assay in cells transfected with TTK plasmids. Results are shown as mean + SD from three independent experiments. Student’s t -test showed that over-expression of TTK significantly increased HR repair efficiency (P<0.05). BRCA1 SMARTpool siRNA was used as a positive control. Western blots demonstrating effective knockdown are shown to the bottom.

Article Snippet: BRCA1 (D-9) monoclonal and TTK polyclonal antibodies were purchased from Santa Cruz (SC-6954, 1:1000) and Cell Signaling (#3255, 1:1000), respectively.

Techniques: Infection, Control, Knockdown, Microarray, Modification, Plasmid Preparation, Electroporation, Flow Cytometry, Colony Assay, Gene Expression, Transfection, Over Expression, Positive Control, Western Blot

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 expression is up-regulated in osteosarcoma cells. (A) , mRNA levels of NRP2 in normal osteoblast (NHOst) and osteosarcoma cell lines (Saos-LM7, 143B, 143.98.2, MG-63, MNNG/HOS, Saos-2, and U2-OS) were determined by real time PCR. (B) , the protein levels of NRP2 in NHOst and seven osteosarcoma cell lines were detected by western blot using NRP2 antibody. The relative protein levels were determined by densitometry and normalized with β-actin level. (C) , Kaplan-Meier survival curves of disease–specific mortality for patients whose osteosarcoma expressed or didn’t express NRP2. The log-rank test was used to compare differences between two groups. NRP2 expression was predictive of poor overall survival. Significant difference is indicated by (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 knockdown inhibited both in vitro and in vivo tumor growth. (A & B) , NRP2 expression was knocked down by NRP2 shRNA in 143B cells. Knockdown efficiency was determined by real-time PCR (A) and Western blotting (B) . (C) , By MTT assay, NRP2 knockdown suppressed anchorage-dependent growth of osteosarcoma 143B cells. (D) Soft agar assay. NRP2 knockdown did not reduce the number of colony formed by 143B cells. 143B cells transfected with ShNRP2 formed smaller colony than control vector transfected cells as shown in the representative images of soft agar (insert). (E) & (F) NRP2 knockdown inhibited the in vivo tumor growth in xenograft nude mice model. 1 × 10 6 143B cells were inoculated in NCR nu-nu nude mice. Tumor size was measured every 3 days and a tumor growth curve was created (E) . Points, mean tumor volume; Bars, SEM. (F) , Representative picture of tumor harvested at day 21. (G) , the knockdown of NRP2 expression in tumor samples was confirmed by immunofluorescence staining using NRP2 antibody. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean value; Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, In Vitro, In Vivo, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Soft Agar Assay, Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: Knockdown of NRP2 inhibited the tumor invasion, migration and lung metastasis of osteosarcoma. (A) , Migration assay. The BD chamber system without Matrigel coating was used to evaluate the migration of shNRP2 and control vector transfected osteosarcoma 143B cells. The migration of 143B cells was significantly inhibited by NRP2 knockdown. (B) , Matrigel invasion assay was performed in BD chamber system coated with Matrigel, using shNRP2 and control vector transfected osteosarcoma 143B cells. There were less NRP2 depleted 143B cells invaded through the matrigel coated porous membrane. (C) , Knockdown of NRP2 in 143B cells reduced the lung metastasis of osteosarcoma in an orthotopic lung metastasis mouse model. Mouse lungs were fixed in Bouin’s solution, and the number of lung surface metastatic nodules was counted and graphed. Each group contained 10 mice and the experiment was repeated 3 times (D) , Representative photographs of lungs with metastatic nodules of osteosarcoma. (E) , NRP2 knockdown significantly reduced both 143B and Saos-2 cells adherence to the endothelial monolayer. The mean cell number was calculated from 10 fields (×100). (F) , Representative images of GFP transfected tumor cells adhering to the endothelial monolayer. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, Migration, Control, Plasmid Preparation, Transfection, Invasion Assay, Membrane

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: Knockdown of NRP2 resulted in decreased blood vessel density of OS in vivo . (A) , Blood vessels (top panel) and capillaries (bottom panel) in tumor samples were visualized by immunohistochemistry with CD31 antibody. Tumor cell nuclear was stained with DAPI. Number of blood vessels per field (100×) was calculated and graphed (Right). Column, mean number of blood vessel per field (x100); Error bars, SEM. (B & C) , Matrigel tube formation assay: no difference was found in the number of tubules formed by HUVEC in conditioned medium from shNRP2 OS cells and shRNA control cells. (B) Representative photographs of tubules formed by HUVEC cells on Matrigel. (C) The tubular number was calculated and graphed, Column: mean number of tubules; Error bars: SEM. (D) , tumor-endothelial co-culture tube formation assay: The HUVEC cells were stained with CellTracker Red CMTPX dye and the tumor cells were transfected with shNRP2 vector with GFP expression. Left panel: tubules formed by HUVEC (red); middle panel: tumor cells (green) attached on tubules; right panel: merged image (right) of HEVEC tubules (red) and attached tumor cells (green). NRP2 depleted tumor cells sustained distinct morphologic changes compared to control cells (bottom panel).

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, In Vivo, Immunohistochemistry, Staining, Tube Formation Assay, shRNA, Control, Co-Culture Assay, Transfection, Plasmid Preparation, Expressing

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 knockdown suppressed endothelial recruitment by osteosarcoma cells. (A) , in a trans-well co-culture migration model, less endothelial cells migrated through the porous insert membrane of the Chamber in shNRP2 group compared to control group. shNRP2 depleted osteosarcoma cells vs control cells were seeded in the lower chamber. HUVECs were seeded in the top insert chamber. Insert picture: Representative photograph of migrated HUVECs. (B) , in a trans-well co-culture invasion model, less endothelial cells invaded through the matrigel coated porous insert membrane of the Chamber in shNRP2 group compared to control group. shNRP2 depleted osteosarcoma cells vs control cells were seeded in the lower chamber. HUVECs were seeded in the top insert chamber. Insert picture: Representative photograph of invaded HUVECs. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean migrated/invaded cells per field (40×); Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, Co-Culture Assay, Migration, Membrane, Control

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 expression is regulated by Wnt signaling pathway. A , Genechip® microarray showed the down-regulated expression of NRP2 in Wnt antagonist sLRP5 transfected osteosarcoma Saos-2 cells. VEGF expression is intact. Real time PCR (B) and western blot (C) with accompanying densitometric assay (D) confirmed the down-regulated mRNA and protein level of NRP2 in Wnt antagonist sLRP5 transfected Saos-2 cells, while VEGF level remains intact. E , western blot and accompanying densitometry showed the down-regulated NRP2 expression in additional Wnt antagonist DKK3 transfected Saos-2 cells, 143B cells and U2-OS cells, and Wif-1 transfected 143B cells. F , Chromatin immunoprecipitation (ChIP) assay verified the binding of TCF4 on the five binding sites in NRP2 promoter region. G , schematic representation of the binding sites of TCF/LEF on the 3-kb promoter region of NRP2 genes.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Binding Assay

Journal: International journal of molecular sciences

Article Title: Yth m 6 A RNA-Binding Protein 1 Regulates Osteogenesis of MC3T3-E1 Cells under Hypoxia via Translational Control of Thrombospondin-1.

doi: 10.3390/ijms24021741

Figure Lengend Snippet: Figure 3. YTHDF1 mediates THBS1 mRNA stability via m6A modification. (A) Overlap of YTHDF1-binding peaks and m6A-containing peaks obtained from microarray data available at https://share.weiyun.com/xcB3waFn, go biological process enrichment analysis of the peaks over- lap were performed using DAVID bioinformatics database. (B) SRAMP program predicted m6A sites in THBS1 mRNA. H: highly putative predicted m6A sites; V–H: very highly putative predicted m6A sites. (C) Prediction of binding sites between YTHDF1 protein and THBS1 mRNA by PRI- dictor. (D) expression of THBS1 transfected with silenced YTHDF1 under normoxia and hypoxia was detected using Western blot and qRT-PCR. (E) RNA stability assays showed THBS1 mRNA of chondrocytes transfected with silenced YTHDF1. (F) The expression and location of YTHDF1 and THBS1 were measured by Immunofluorescence (IF) assays in MC3T3-E1 cells cultured for 24 h under normoxia (21% O2) or hypoxia (1% O2). Scale bar, 200 µm, n = 3. Results are presented as mean ± SD. The p values were calculated by one-way ANOVA followed by Tukey’s test (* p < 0.05, *** p < 0.001, compared with the control group). YTHDF1, Yth m6A RNA-binding protein 1; THBS1, thrombospondin-1; Act D, actinomycin D.

Article Snippet: Afterward, the samples were blocked with 5% bovine serum albumin for 1 h and probed by specific primary antibodies for YTHDF1 (1:100; Proteintech, Wuhan, China), THBS1(1:100; Proteintech, Wuhan, China) at 4 ◦C overnight after treatment.

Techniques: Binding Assay, Microarray, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Cell Culture, Control, RNA Binding Assay

Journal: International journal of molecular sciences

Article Title: Yth m 6 A RNA-Binding Protein 1 Regulates Osteogenesis of MC3T3-E1 Cells under Hypoxia via Translational Control of Thrombospondin-1.

doi: 10.3390/ijms24021741

Figure Lengend Snippet: Figure 4. THBS1 promotes MC3T3-E1 osteogenic differentiation under hypoxic conditions. (A,B) Pro- tein and mRNA expression of THBS1 in MC3T3-E1 cells transfected with silenced THBS1 were detected after 48 h cultured under hypoxia (1% O2). (C,D) Protein and mRNA expression levels of ALP, RUNX2, and COL1 after transfected with si-Thbs1 were detected using qRT-PCR and Western blot in comparison with si-NC under normoxia or hypoxia. (E,F) ALP and ARS staining show the inhibitory effect of si-Thbs1 knocking down THBS1 on differentiation of MC3T3-E1 osteoblasts cultured in the osteogenic

Article Snippet: Afterward, the samples were blocked with 5% bovine serum albumin for 1 h and probed by specific primary antibodies for YTHDF1 (1:100; Proteintech, Wuhan, China), THBS1(1:100; Proteintech, Wuhan, China) at 4 ◦C overnight after treatment.

Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Comparison, Staining

Journal: International journal of molecular sciences

Article Title: Yth m 6 A RNA-Binding Protein 1 Regulates Osteogenesis of MC3T3-E1 Cells under Hypoxia via Translational Control of Thrombospondin-1.

doi: 10.3390/ijms24021741

Figure Lengend Snippet: Figure 5. Overexpression of YTHDF1 promotes osteogenic differentiation of MC3T3-E1 cells via YTHDF1/THBS1 pathway under hypoxic conditions. (A,B) YTHDF1 mRNA and protein expression determined through qRT-PCR and Western blot after 48 h transfection of siRNAs or negative control in MC3T3-E1 cells. (C) mRNA expression of YTHDF1 and THBS1 were examined after overexpression of YTHDF1 under normoxia or hypoxia. (D) The protein expression levels of ALP, RUNX2, and COL1 after transfection with overexpressed YTHDF1 were detected by Western blot, compared with NC-oe under normoxia or hypoxia. (E) The protein expression levels of ALP, RUNX2, and COL1 after co-transfection of si-Thbs1 with Ythdf1-oe under normoxia or hypoxia. (F) ALP staining showed that overexpression of YTHDF1 promoted differentiation of MC3T3-E1 osteoblasts in both normoxic and hypoxic cultures, while the knockdown of THBS1 showed a slight regression of the osteogenic ability promoted by Ythdf1-oe. Scale bar, 200 µm, n = 3. Results are presented as mean ± SD. The p values were calculated by one-way ANOVA followed by Tukey’s test (* p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group). YTHDF1, Yth m6A RNA-binding protein 1; THBS1, thrombospondin-1; ALP, alkaline phosphatase; RUNX2, runt-related transcription factor 2; COL1, type 1 collagen.

Article Snippet: Afterward, the samples were blocked with 5% bovine serum albumin for 1 h and probed by specific primary antibodies for YTHDF1 (1:100; Proteintech, Wuhan, China), THBS1(1:100; Proteintech, Wuhan, China) at 4 ◦C overnight after treatment.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Cotransfection, Staining, Knockdown, Control, RNA Binding Assay

Journal: International journal of molecular sciences

Article Title: Yth m 6 A RNA-Binding Protein 1 Regulates Osteogenesis of MC3T3-E1 Cells under Hypoxia via Translational Control of Thrombospondin-1.

doi: 10.3390/ijms24021741

Figure Lengend Snippet: Figure 6. The role of the RNA-binding protein of N6-methyladenosine methylation YTHDF1 in a hypoxia-induced MC3T3-E1 cell model of osteoblasts. Hypoxia inhibited osteogenic differentiation and promoted the expression of YTHDF1 and THBS1, and the depletion of either YTHDF1 or THBS1 exacerbated the osteogenic inhibition caused by hypoxia, respectively. The increased expression of YTHDF1 under hypoxia enhanced the translation of THBS1 under hypoxia by stabilizing the mRNA transcript of THBS1. Inflammation-induced hypoxia aggravates bone resorption in peri-implantitis, while hypoxia-activated YTHDF1 with THBS1 promotes osteogenesis to prevent bone resorption. YTHDF1, Yth m6A RNA-binding protein 1; THBS1, thrombospondin-1.

Article Snippet: Afterward, the samples were blocked with 5% bovine serum albumin for 1 h and probed by specific primary antibodies for YTHDF1 (1:100; Proteintech, Wuhan, China), THBS1(1:100; Proteintech, Wuhan, China) at 4 ◦C overnight after treatment.

Techniques: RNA Binding Assay, Methylation, Expressing, Inhibition