Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 1 Regulation of PKN kinase activity. IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. B, The PKN1 TM mutant S916A has reduced kinase activity toward multiple substrates. C, Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. D and E, The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 3 Torin and rapamycin sensitivity of PKN, AKT, and PKCα. A, Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 h, and analyzed by using pan- and phosphosite-specific antibodies. B, Cells were treated with torin and rapamycin during a time course up to 24 h and subsequently analyzed by using pan- and phosphosite- specific antibodies

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 2 PKN contains a TM phosphorylated by a torin- sensitive kinase. A, Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). B, Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including nonphospho-TM peptide during the antibody incubation reduces the detection of non- phosphorylated PKN. C, IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 4 Cell motility functions of PKN. A, Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. B, Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. C, Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. D, Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. E, Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (****P < 0.0001). G, Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 5 Analysis of PKN isoform expression in human prostate cancer. A, Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. B, PKN1 and PKN2 expression (using microarray data from reference 47) in normal prostate, primary tumor, and metastases. C, RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT, and select mTOR components. **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 6 Pkn2 is required for embryonic development. A, Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β- galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0 mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2 mm, 0.5 mm, 1.0 mm. B, Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75, and E9.5. Scale bars 0.2 mm (upper four panels) 1.0 mm. C, Whole mount images of wild-type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2 mm

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 7 Analysis of PKN1 overexpression in prostate. A, Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). B-E, H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1;Tg-PKN1, 41 weeks; Tg-AKT1;Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP;Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP;Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Figure S3)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Over Expression, Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 8 Analysis of PKNs in Pten null prostate tumors. H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200× magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r;Pkn2r/r, 26 and 45 weeks. D, The images of invasive cancer (left and middle) are from 53-week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53-week animal. Lower magnification views of the same samples are also provided (Supplemental Figure S4)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining

Journal: Oncogene

Article Title: Stat3 Contributes to Cancer Progression by Regulating Jab1/Csn5 Expression

doi: 10.1038/onc.2016.271

Figure Lengend Snippet: (A) Overall p-Stat3, T-Stat3, and Jab1 immunoreactivity in nonneoplastic tissues (left) was lower than that in NPC (right). The percentages of nonneoplastic nasopharyngeal tissues or NPC specimens with p-Stat3, T-Stat3, and Jab1 expression are shown at right. Original magnification, ×200; insets, ×400. (B) Nuclear p-Stat3 expression was associated with nuclear Jab1 in NPC tissues. R and P values were obtained using the Spearman test. (C–E) Jab1 and Stat3 gene expression in normal nasopharynx and NPC using the Oncomine gene expression tool ( https://www.oncomine.com ). The clinical data were downloaded from Oncomine Data Portal; C, Heat-map of Stat3 and Jab1 gene expression. Jab1 gene (D) and Stat3 gene (E) expression in normal nasopharynx and NPC. (F and G) Kaplan-Meier analyses of the association between p-Stat3 or T-Stat3 protein expression and survival (F) and the association between combined Stat3 and Jab1 protein expression (G) and survival.

Article Snippet: Antibodies to the following proteins were used: Jab1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-13157); PARP (BD Pharmingen, San Diego, CA, Cat#556494); p-Stat3 and T-Stat3 (Cell Signaling Technology, Beverly, MA, Cat#9145 and Cat#12640); Myc-tag (Roche Applied Biosciences, Indianapolis, IN, Cat#11667149001); and Flag and β-actin (Sigma-Aldrich, St. Louis, MO, Cat#F3165 and Cat#A5441).

Techniques: Expressing, Gene Expression

Journal: Oncogene

Article Title: Stat3 Contributes to Cancer Progression by Regulating Jab1/Csn5 Expression

doi: 10.1038/onc.2016.271

Figure Lengend Snippet: (A) Whole-cell lysates were prepared from the cells as indicated. β-actin was used as a control for protein loading and integrity. The relative p-Stat3, T-Stat3, and Jab1 intensities for six samples are shown. (B) Ectopic Stat3 increased Jab1 expression in NPC cells. NPC cells were transfected by incubation with the Flag-Stat3 plasmid for 48 hours. The cells were then lysed and subjected to Western blotting for detection of Flag and Jab1 protein levels. (C) Knockdown of Stat3 downregulated endogenous Stat3 levels in NPC cell lines. Lysates were prepared from Stat3 siRNA-infected cells. (D) NPC cells were transfected with increasing [10 pM (+) and 40 pM (++)] doses of Stat3 siRNA for 48 hours, and Stat3 and Jab1 RNA levels were examined via quantitative PCR. (E) NPC cells with stable knockdown of Stat3 were established, and two clones for each cell line were selected for Stat3 and Jab1 detection. (F) NPC cells were exposed to CYT387 at the indicated concentration for 48 ho, and then were detected for p-Stat3 and Jab1 protein expression by western blotting. (G) Progressive deletions of the 5′ region of the Jab1 promoter in luciferase (Luc) constructs were transfected into NP460 and CNE1 cells and subjected to luciferase reporter assays. Promoter activity was higher in CNE1 cells than in NP460 cells. Deletion of the region −472 to −344 containing Stat3 binding site (−446/−423) resulted in a loss of promoter activity in CNE1 cells. (H) ChIP assay was carried out using Stat3 and immunoglobulin G (IgG) antibodies, and the extracted DNA was amplified by real-time PCR. The data are the means with standard deviations for three independent experiments. ** P < 0.01. Cont, Control.

Article Snippet: Antibodies to the following proteins were used: Jab1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-13157); PARP (BD Pharmingen, San Diego, CA, Cat#556494); p-Stat3 and T-Stat3 (Cell Signaling Technology, Beverly, MA, Cat#9145 and Cat#12640); Myc-tag (Roche Applied Biosciences, Indianapolis, IN, Cat#11667149001); and Flag and β-actin (Sigma-Aldrich, St. Louis, MO, Cat#F3165 and Cat#A5441).

Techniques: Control, Expressing, Transfection, Incubation, Plasmid Preparation, Western Blot, Knockdown, Infection, Real-time Polymerase Chain Reaction, Clone Assay, Concentration Assay, Luciferase, Construct, Activity Assay, Binding Assay, Amplification

Journal: Oncogene

Article Title: Stat3 Contributes to Cancer Progression by Regulating Jab1/Csn5 Expression

doi: 10.1038/onc.2016.271

Figure Lengend Snippet: (A) NPC cells were transfected with Stat3 siRNA for 48 hours and C666.1 cells stably infected with Stat3 shRNA (sh-Stat3)- or control shRNA (sh-Cont) carrying lentivirus, and cell growth was determined via an MTT assay. (B) Representative results of colony formation assays with NPC cells treated with control siRNA or 10 pM (+) or 40 pM (++) Stat3 siRNA. The relative numbers of colonies are shown at the right panel. (C) NPC cells transfected with Stat3 siRNA or Jab1 siRNA were exposed to invasion chamber assay. Stat3 and Jab1 mRNA levels were determined by RT-QPCR (Right, top). Matrigel membranes containing invading cells were observed via optical microscopy (Left), and the cells were counted (Right, bottom). The number of invading cells from each cell population was quantified. Cont-si, Control siRNA; Jab1-si, Jab1 siRNA. The data are means with standard deviations for three independent experiments. ** P < 0.01.

Article Snippet: Antibodies to the following proteins were used: Jab1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-13157); PARP (BD Pharmingen, San Diego, CA, Cat#556494); p-Stat3 and T-Stat3 (Cell Signaling Technology, Beverly, MA, Cat#9145 and Cat#12640); Myc-tag (Roche Applied Biosciences, Indianapolis, IN, Cat#11667149001); and Flag and β-actin (Sigma-Aldrich, St. Louis, MO, Cat#F3165 and Cat#A5441).

Techniques: Transfection, Stable Transfection, Infection, shRNA, Control, MTT Assay, Invasion Chamber Assay, Quantitative RT-PCR, Microscopy

Journal: Oncogene

Article Title: Stat3 Contributes to Cancer Progression by Regulating Jab1/Csn5 Expression

doi: 10.1038/onc.2016.271

Figure Lengend Snippet: (A–D) Stat3 expression levels and cisplatin (CP) responses of CNE2 and HONE1 cells transiently transfected with Stat3 siRNA (Stat3-si) or scrambled control siRNA (Cont-si) and of C666.1 cells stably infected with Stat3 shRNA (sh-Stat3)- or control shRNA (sh-Cont)-carrying lentivirus as determined by MTT assay (A), colony formation assay (B), annexin-V/PI staining (C), and PARP cleavage in CNE2 cells (D). (E, F) CNE2 cells stably expressing sh-Stat3 were transfected with pcDNA or Myc-Jab1 plasmid DNA. (E) Western blot analyses demonstrated the effective knockdown and ectopic expression. (F) Colonies were stained with crystal violet 10 days after CP exposure. The data are means with standard deviations for three independent experiments. ** P < 0.01. OD, optical density.

Article Snippet: Antibodies to the following proteins were used: Jab1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-13157); PARP (BD Pharmingen, San Diego, CA, Cat#556494); p-Stat3 and T-Stat3 (Cell Signaling Technology, Beverly, MA, Cat#9145 and Cat#12640); Myc-tag (Roche Applied Biosciences, Indianapolis, IN, Cat#11667149001); and Flag and β-actin (Sigma-Aldrich, St. Louis, MO, Cat#F3165 and Cat#A5441).

Techniques: Expressing, Transfection, Control, Stable Transfection, Infection, shRNA, MTT Assay, Colony Assay, Staining, Plasmid Preparation, Western Blot, Knockdown

Journal: Oncogene

Article Title: Stat3 Contributes to Cancer Progression by Regulating Jab1/Csn5 Expression

doi: 10.1038/onc.2016.271

Figure Lengend Snippet: (A–C) Representative photographs of harvested tumors (top) and the corresponding tumor growth curves (bottom) are shown. Tumor weights measured at the indicated times are shown at right. Female nude mice bearing xenograft tumors derived from CNE2 cells (A), HONE1 cells (B), or C666.1 cells (C) with stable Stat3 knockdown were intraperitoneally injected with phosphate-buffered saline or cisplatin (CP) at 5 mg/kg of body weight once every 2 days. (D) At the end of the experiments, the mice were humanely killed, and the tumors were excised and weighed. The data are means with standard errors. (E) Representative T-Stat3, and Jab1 expression in tissues from the same mice. Correlations of Stat3 and Jab1 staining scores are shown at right. IHC, immunohistochemistry. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies to the following proteins were used: Jab1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-13157); PARP (BD Pharmingen, San Diego, CA, Cat#556494); p-Stat3 and T-Stat3 (Cell Signaling Technology, Beverly, MA, Cat#9145 and Cat#12640); Myc-tag (Roche Applied Biosciences, Indianapolis, IN, Cat#11667149001); and Flag and β-actin (Sigma-Aldrich, St. Louis, MO, Cat#F3165 and Cat#A5441).

Techniques: Derivative Assay, Knockdown, Injection, Saline, Expressing, Staining, Immunohistochemistry

Journal: Oncogene

Article Title: Stat3 Contributes to Cancer Progression by Regulating Jab1/Csn5 Expression

doi: 10.1038/onc.2016.271

Figure Lengend Snippet: In vivo cDNA microarray data and Kaplan-Meier plots were used to assess correlations between Stat3 and Jab1 expression and patient survival. (A–C) Higher expression of Stat3 and Jab1 is associated with worse survival in patients with colon cancer (A), breast cancer (B), and glioblastoma (C). (D) Overview of the two genes across datasets.

Article Snippet: Antibodies to the following proteins were used: Jab1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-13157); PARP (BD Pharmingen, San Diego, CA, Cat#556494); p-Stat3 and T-Stat3 (Cell Signaling Technology, Beverly, MA, Cat#9145 and Cat#12640); Myc-tag (Roche Applied Biosciences, Indianapolis, IN, Cat#11667149001); and Flag and β-actin (Sigma-Aldrich, St. Louis, MO, Cat#F3165 and Cat#A5441).

Techniques: In Vivo, Microarray, Expressing

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: (A) The mRNA expression level of GPR116 in colon cancer and matched normal colon tissue derived from the Renji cohort was detected by real-time quantitative PCR. Error bars in the scatter plots represent SE. (B) Western blots showed GPR116 protein level in six paired colon cancer tissues (T) and adjacent normal colonic tissue (N) of patients from the Renji cohort and the relative GPR116 protein expression. GPR116 expression in Skrzypczak Colorectal (C) Skrzypczak Colorectal 2 (D) and Gaedcke Colorectal (E) grouped by normal colorectal tissue (1) and colorectal carcinoma (2) in Oncomine Cancer Microarray database. CRC: colorectal carcinoma; GPR116: G protein-coupled receptor 116; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SE: standard error.

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

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

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: (A) GPR116 protein level was measured by immunohistochemical analysis in normal colon and colon cancer tissue with different staining intensities. Original magnification ×100 (bar=200μm); 200 (bar=100μm); 400 (bar=50μm). (B) The numbers of different immunohistochemical grade expression in normal colon and colon cancer tissue. NC: normal colon.

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: Association between GPR116 expression and clinicopathologic features in patients with colorectal carcinoma

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Expressing

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: (A) Overall survival analysis of 90 CRC patients with different GPR116 protein expression in the Renji cohort. (B-D) The association of GPR116 expression and patient survival was conducted in GSE14333, GSE17536, and GSE33113 datasets, respectively. (E-F) Comparisons of overall survival between the lower GPR116 expression group and the higher GPR116 expression group in the early TNM stage (I-II) cohort and in the advanced TNM stage (III-IV) cohort. (G-H) Comparisons of overall survival between the lower GPR116 expression group and the higher GPR116 expression group in patients with or without lymph node metastasis. p -values were calculated by Log-rank test. TNM: tumor-node –metastasis.

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Expressing

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: Univariate and multivariate analysis of prognostic parameters for survival in patients with colorectal carcinoma

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Expressing

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: (A-B) The forest plot showed the correlation between CRC patient overall survival and GPR116 expression as well as other clinical characteristics by using univariable and multivariable analysis. (C) ROC curve analysis according to GPR116 expression by RT-PCR. (D) ROC curve analysis according to GPR116 expression by IHC. ROC: receiver operating characteristic; IHC: immunohistochemical.

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: (A-D) GSEA revealed that “Regulation_Of_Cell_Proliferation”, “Gildea_Metastasis”, “Gotzmann_Epithelial_ To_Mesenchymal_Transiton_Up” and “Alonso_Metastasis_ EMT_Up” pathway were enriched in GPR116-high expression group compared with GPR116-low expression group. The enrichment score (ES, green line) means the degree to which the gene set is overrepresented at the top or bottom of the ranked list of genes. EMT: epithelial-mesenchymal transition.

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Expressing

Journal: Oncotarget

Article Title: High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma

doi: 10.18632/oncotarget.18203

Figure Lengend Snippet: (A-B) GPR116 expression was measured in normal colonic cell line FHC and four colon cancer cell lines by real-time PCR and western bolt. (C-D) The cell viability was measured by CCK-8 assays at various time points in HCT116 and LOVO cell. (* p <0.05, versus control siRNA). (E-F) The cell invasion ability was measured by transwell chamber assay in HCT116 and LOVO cell; the results of transwell chamber assay were quantitated by counting invasive cells in five randomly selected high-power fields for three replicates (magnification, ×200). Results shown are the mean ± SD of triplicate determinations from three independent experiments.

Article Snippet: After being blocked with 10% goat serum for 30 minutes, the section was incubated with rabbit GPR116 antibody (1:100, Proteintech Group, USA) overnight at 4°C, followed by a peroxidase-labeled secondary antibody.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Control, Transwell Chamber Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: DNA sequences of the primers used for traditional and real time RT-PCR

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Amplification

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Microarray, Expressing, Sequencing, Ubiquitin Proteomics, Binding Assay, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Induction of chemokine gene expression by LCN2 in astrocytes, microglia, endothelial cells, and neuron cells. Astrocytes (A and E), microglia (B and E), bEnd.3 endothelial cells (C), and neuron cells (D) were treated with the recombinant LCN2 protein (10 μg/ml) for 8 h, and the total RNA was isolated for traditional RT-PCR or real time PCR. The cells were also treated for 8 h with LPS (100 ng/ml), TNF-α (10 ng/ml), or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for comparison purposes. The mRNA levels of chemokines (CCL4, CCL20, CXCL2, and CXCL10) and other inflammatory genes (IL-6, COX-2, iNOS, and PIAS3) were determined by traditional RT-PCR (A–D) or real time PCR (E). β-Actin or GAPDH was used as an internal control. The results are one representative of more than three independent experiments (A–D) or means ± S.D. (n = 3) (E).

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Gene Expression, Recombinant, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: An increase of CXCL10 expression by LCN2 in astrocytes. Astrocytes were incubated with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h. The amounts of CXCL10 protein in the culture media were measured by specific ELISA (A). The results are means ± S.D. (n = 3). *, p < 0.05 compared with the untreated control. The stable overexpression or knockdown of lcn2 expression was achieved by transfection with sense or antisense lcn2 cDNA in C6 rat glioma cells. The increased or decreased lcn2 expression in the stable transfectants (S3, lcn2 sense transfectant; AS7, lcn2 antisense transfectant) compared with cells transfected with an empty vector (V2) was confirmed by RT-PCR (B). Changes in the CXCL10 mRNA levels in the stable transfectants were also assessed by RT-PCR (C). β-Actin was used as an internal control. The results are one representative of more than three independent experiments.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Expressing, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Astrocyte-derived CXCL10 promoted the migration of astrocytes. Astrocytes (1 × 104 cells/upper well) were exposed to LCN2 (10 μg/ml)-stimulated ACM or the recombinant CXCL10 protein (10 ng/ml) in the presence or absence of CXCL10 neutralizing antibody (CXCL10 Ab; 10 ng/ml) as indicated. ACM-None, untreated ACM; ACM-LCN2, LCN2-treated ACM (see “Experimental Procedures” for the preparation of ACM). After treatment for the indicated time periods, either wound healing assay (A) or the Boyden chamber assay (B) was performed to evaluate cell migration. A representative microscopic image for each condition was shown (magnification, ×100) (upper). The quantification of cell migration was done by either measuring the degree of wound closure (wound healing assay) or enumerating the migrated cells (Boyden chamber assay) as described under “Experimental Procedures” (lower). The results are means ± S.D. (n = 3). *, p < 0.05 compared with ACM-None at the same time point; **, p < 0.05 compared with ACM-LCN2 at the same time point; #, p < 0.05 compared with the untreated control (None) at the same time point. For the checkerboard analysis, migration of astrocytes (2 × 104 cells/upper well) in response to the indicated concentrations of ACM-LCN2 (C) and the recombinant CXCL10 protein (D) placed in upper and/or lower well was determined using the Boyden chamber assay. The quantification of cell migration was done by enumerating the migrated cells after 48 h as described under “Experimental Procedures.” The results are the means ± S.D. (n = 3). *, p < 0.05 between the treatments indicated.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Derivative Assay, Migration, Recombinant, Wound Healing Assay, Boyden Chamber Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Checkerboard analysis for promigratory effects of recombinant CXCL10 protein in astrocytes The recombinant CXCL10 protein was added to the upper and/or lower wells of the Boyden chambers for the checkerboard analysis. After cells were incubated at 37 °C under 5% CO 2 for 48 h, cell migration was assessed as described under “Experimental Procedures.” NT, not tested.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Recombinant, Incubation, Migration

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: The effects of LCN2 protein expressed in mammalian cells (mLCN2) on the chemokine gene expression and cell migration in astrocytes. Astrocytes were treated with the NSO murine melanoma cell-derived mouse LCN2 protein (10 μg/ml; mLCN2) for 8 h, and the total RNA was isolated for traditional RT-PCR. The cells were also treated for 8 h with LPS (100 ng/ml) plus IFN-γ (50 units/ml) for comparison purposes. The mRNA levels of CXCL10 and PIAS3 were determined by traditional RT-PCR (A). β-Actin was used as an internal control. The results are one representative of more than three independent experiments. Astrocytes (1 × 104 cells/upper well) were exposed to the melanoma cell-expressed LCN2 protein (10 μg/ml; mLCN2)-stimulated astrocyte-conditioned media (ACM-mLCN2). Astrocytes placed in the Boyden chambers were then incubated at 37 °C for 24–72 h to evaluate cell migration (B). A representative microscopic image for each condition was shown (magnification, ×100) (upper). ACM-None, untreated ACM; ACM-mLCN2, mLCN2-treated ACM. The quantification of cell migration was done by enumerating the migrated cells as described under “Experimental Procedures” (lower). The results are the means ± S.D. (n = 3). *, p < 0.05 compared with ACM-None at the same time point.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Gene Expression, Migration, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Comparison, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Astrocyte-derived CXCL10 promoted the migration of microglia and neuron cells. Microglia (1 × 104 cells/upper well) (A) or neuron cells (1 × 104 cells/upper well) (B) were exposed to LCN2 (10 μg/ml)-stimulated ACM or the recombinant CXCL10 protein (10 ng/ml) in the presence or absence of CXCL10 neutralizing antibody (10 ng/ml) as indicated. Microglia or neuron cells placed in the Boyden chambers were incubated at 37 °C for 12–48 or 18–48 h, respectively, to evaluate cell migration. The GST protein (10 μg/ml) was used as a control for the recombinant LCN2 protein. A representative microscopic image for each condition is shown (magnification, ×100) (upper). ACM-None, untreated ACM; ACM-LCN2, LCN2-treated ACM. The quantification of cell migration was done by enumerating the migrated cells as described under “Experimental Procedures” (lower). The results are the means ± S.D. (n = 3). *, p < 0.05 compared with ACM-None at the same time point; **, p < 0.05 compared with ACM-LCN2 at the same time point; #, p < 0.05 compared with the untreated control (None) at the same time point.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Derivative Assay, Migration, Recombinant, Incubation, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: JAK2/STAT3 and IKK/NF-κB mediated LCN2 up-regulation of CXCL10 and GFAP expression in astrocytes. Astrocytes were pretreated with the recombinant LCN2 protein (10 μg/ml) for 24 h prior to the treatment with phorbol 12-myristate 13-acetate (100 μg/ml), ATP (3 mm), or IFN-γ (50 units/ml) for 30 min. Astrocytes were also exposed to phorbol 12-myristate 13-acetate (PMA), ATP, or IFN-γ for 30 min without LCN2 pretreatment (A). Alternatively, astrocytes were treated with LCN2 for 0.5–24 h for the time kinetics analysis (B). The levels of phosphorylated STAT3 (pSTAT3 at Ser727 or Tyr705) or total STAT3 protein were then evaluated by Western blot analysis. The results are one representative of more than three independent experiments. Alternatively, astrocytes were pretreated with AG490 (JAK2/STAT3-specific inhibitor, 50 μm) or piceatannol (JAK1/STAT1-specific inhibitor, 50 μm) for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h. The secreted CXCL10 protein was measured by specific ELISA (C). The results are the means ± S.D. (n = 3). *, p < 0.001 compared with the treatment without inhibitors. Astrocytes were pretreated with AG490 for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) for 24 h. The expression of GFAP protein levels was assessed by Western blot analysis, respectively (D). After astrocytes were treated with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 1 h, an EMSA analysis of the nuclear extracts was conducted by using a 32P-labeled NF-κB oligonucleotide probe (E). Binding specificity was determined by the supershift assay using antibody against p65 (p65 Ab) or its coincubation with an unlabeled probe containing the NF-κB binding sequence (cold probe) to compete with the labeled oligonucleotide. The results are one representative of more than three independent experiments. Primary astrocytes were pretreated with pyrrolidine dithiocarbamate (PTDC, NF-κB-specific inhibitor; 0–10 μm) for 30 min prior to their treatment with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h (F). The concentration of nitrite in the culture media was measured by the Griess reagent. The results are the means ± S.D. (n = 3). *, p < 0.001 compared with the LCN2 or LPS/IFN-γ treatment alone. The astrocytes were pretreated with SC-514 (IKK-specific inhibitor, 10 μm) for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) for 8–24 h. The expression of GFAP at mRNA or protein levels after 8 or 24 h was then assessed by RT-PCR or Western blot analysis, respectively (G).

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Expressing, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, Sequencing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Role of LCN2 in astrocyte migration and CXCL10 induction in cortical stab wound injury model. Cortical stab wound injury was performed with LCN2 wild-type (LCN2+/+) or LCN2-deficient mice (LCN2−/−) (A, upper). At 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against GFAP. The asterisk indicates stab wound injury site. The boxes indicate 200-μm × 200-μm squares placed for cell counting. Immunohistochemistry results showed that GFAP-positive cells in the peri-injury region were observed in both LCN2+/+ and LCN2−/− mice. A significant decrease in the number of GFAP-positive cells was observed in the immediate vicinity of injury site in LCN2−/− mice (A, lower panel). The results are one representative of more than three independent experiments. Scale bars, 200 μm. The values are the means ± S.D. from three different animals and six independent sections/animal. *, p < 0.05 compared with LCN2−/− mice in the same counting area; #, p < 0.05 between the values indicated. The mRNA levels of lcn2 (upper panel) and CXCL10 (lower panel) in LCN2+/+ and LCN2−/− mice were examined at 2 days after cortical stab wound injury (B). RNA was isolated from the injury site in the cortex and subjected to real time PCR. The injury-induced CXCL10 expression was completely abrogated in LCN2−/− brain as compared with LCN2+/+ littermates. GAPDH was used as a control in the real time PCR. The results are the means ± S.D. (n = 3). *, p < 0.05 compared with uninjured LCN2+/+ mice; **, p < 0.05 compared with injured LCN2+/+ mice; #, p < 0.05 compared with uninjured LCN2−/− mice.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Migration, Cell Counting, Immunohistochemistry, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Essential role of LCN2 in reactive astrocytosis and CXCL10 induction in LPS-induced mouse neuroinflammation models. LCN2+/+ or LCN2−/− were injected with LPS intracortically (A) or icv (B). After 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against LCN2 (green, upper) or GFAP (red, middle). The nuclei were counterstained with DAPI (blue, lower). The asterisk indicates the injection site. A significant decrease in both LCN2 and GFAP expression was observed in LCN2−/− mice. The results are one representative of more than three independent experiments. Scale bars, 100 μm. Quantification of the GFAP-positive cells was done in the cortex (A, upper graph) or hippocampus (B, upper graph). The values are the means ± S.D. from three different animals and five independent sections per animal. The mRNA levels of lcn2, CXCL10, and GFAP in LCN2+/+ and LCN2−/− mice were examined by real time PCR of cortical tissue around the injection site (A, lower graph) or hippocampus (B, lower graph) at 2 days after intracortical or icv injection with LPS, respectively. LCN2−/− mice exhibited markedly lower levels of CXCL10 and GFAP as compared with LCN2+/+ littermates. *, p < 0.05; compared with wild-type LPS-injected mice (LCN2+/+) at the same inflammation model. At 2 days after icv injection of LCN2+/+ mice with LPS, hippocampus was immunostained with antibodies against LCN2 (green) or GFAP (red) (C). The nuclei were counterstained with DAPI (blue). A merged image is shown in the lower right panel. The arrowheads indicate colocalization of LCN2 and GFAP (yellow). Cell bodies and processes of astrocytes in hippocampus were stained with LCN2 antibody. The results are representative of more than three independent experiments. Scale bars, 20 μm.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Schematic diagram depicting the promotion of CNS cell migration by LCN2-induced CXCL10 (A) and the possible pathway through which LCN2 induces astrocyte migration and morphological changes (B). A, LCN2 up-regulates CXCL10 expression in the multiple cell types in the CNS, such as astrocytes, microglia, neurons, and endothelial cells. Astrocyte-derived CXCL10 acts in a paracrine or autocrine manner to promote cell migration in the inflammatory scene. CXCL10 secreted by other cell types may play a similar role. The lcn2 receptor and CXCL10 receptor (CXCR3) are widely expressed in glia, endothelial cells, and neurons. B, LCN2 up-regulates CXCL10 and GFAP expression in reactive astrocytes through JAK2/STAT3 and NF-κB pathways. Although LCN2-up-regulated CXCL10 promotes cell migration, GFAP induction may lead to morphological changes observed in reactive astrocytosis. Based on the microarray analysis, LCN2 induces the up-regulation of IL-6 and down-regulation of PIAS3, thereby facilitating the STAT3 pathway (dotted line). NO production, downstream of the NF-κB, may cooperate with the STAT3 pathway to induce GFAP expression. NO has been shown to induce GFAP expression in astrocytes (bold dotted line) (54). Moreover, IL-6 previously induced GFAP expression through the STAT3 pathway (96). Other pathways may also participate in astrocyte migration and morphological change under the current conditions.

Article Snippet: The levels of CXCL10 in the culture media were measured by sandwich ELISA using a rat monoclonal anti-mouse CXCL10 antibody as the capture antibody and goat biotinylated polyclonal anti-mouse CXCL10 antibody as a detection antibody (ELISA development reagent; R & D Systems), respectively, as previously described ( 37 ).

Techniques: Migration, Expressing, Derivative Assay, Microarray

Journal: Cell Death & Disease

Article Title: ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer

doi: 10.1038/s41419-022-04862-1

Figure Lengend Snippet: A ICAM-1 expression data in normal colon tissue ( n = 54) and colorectal cancer tissue ( n = 186) was obtained from the Gene Expression Omnibus (GEO) database. B Tissue microarray analysis of ICAM-1 expression in normal ( n = 17) and cancer ( n = 588) type of colorectal tissues. C qRT-PCR was performed to detect the expression of ICAM-1 in various colorectal cancer cell lines and normal colon cell. D , E Kaplan–Meier survival analysis cohorts based on low and high expression of ICAM-1 in colorectal cancer. (TCGA, n = 302). F GSEA (GSE4183) analysis was performed in colorectal cancer that ICAM-1 high expressed for hallmarks of cancer progression. NES normalized enrichment score; FDR q val false discovery rate q -value. G Transwell chamber assays were performed to determine whether expression of ICAM-1 modulates EMT after silencing ICAM-1 using SW-480 cells. H SW-480 cells transfected with sh-CTR and sh-ICAM-1 (3 × 10 6 ) were injected into the cecal wall of athymic BALB/c-nude (8-weeks old) mice ( n = 4). I Representative H&E staining images of lung metastases. And the graph shows the number of lung metastatic lesions in each mouse . J , K qRT-PCR and IHC staining of expression levels for EMT markers and regulators in tumor tissues of sh-CTR and sh-ICAM-1 injected mice. L Tube-forming ability of HUVEC cells was performed by incubation with SW-480 in which ICAM-1 was silenced. Tube formation was assessed after 2 h using light microscopy, and the Image J program was used to analyze tube length. M , N qRT-PCR and IHC staining of expression levels for that angiogenesis-related markers and growth factors in tumor tissues of sh-Ctr and sh-ICAM-1 injected mice. O Representative immunofluorescence image for CD31 (red) and VEGFA (green) in mouse tumors. Scale bar, 100 μm, β-actin and positive control were used as a control for normalization. Data are presented as mean ± SD and analyzed by Student’s t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human p-CMV-ICAM-1 vector was purchased from sinobiological, pc-DNA3-SRC vector was purchased from Addgene, and p-CMV6-c-MET vector was purchased from origene.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Transfection, Injection, Staining, Immunohistochemistry, Incubation, Light Microscopy, Immunofluorescence, Positive Control

Journal: Cell Death & Disease

Article Title: ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer

doi: 10.1038/s41419-022-04862-1

Figure Lengend Snippet: A Kinase assay was performed on SW-480 cells in which ICAM-1 was silenced, and the intensity of each dot was analyzed using the Image J program ( n = 2). B Pearson correlation analysis of ICAM-1 and p-SRC expression levels in TCGA colon cancer patient cohort. C GSEA of SRC oncogene signature gene to ICAM-1 expression in colorectal cancer patients from GSE44076. D qRT-PCR of representative SRC signature genes was performed in SW-480 cells transfected with si-CTR and si-ICAM-1. E SRC kinase assay was detected using luminescence in SW-480 cells with reduced ICAM-1 expression. F , G IHC staining assays and staining intensity score analysis of p-SRC in control and ICAM-1 knockdown mouse tissues. H , I Transwell chamber assay and tube formation assay were performed using HT-29 cells with overexpressed ICAM-1 and SRC inhibitors treatment. Tube formation was assessed after 2 h using light microscopy, and the Image J program was used to analyze tube length. J Western blot on the expression level of EMT and angiogenesis-related regulators in HT-29 cells. K Western blotting in HT-29 cells after treatment with overexpressed ICAM-1 and SRC inhibitors. L – N Transwell chamber assay, tube formation assay, and western blot were performed using HT-29 cells with overexpressed ICAM-1 and STAT3 inhibitors treatment. Tube formation was assessed after 2 h using light microscopy, and the Image J program was used to analyze tube length. O Quantitative real-time PCR analysis of ICAM-1 mRNA expression in HT-29 after treatment with overexpressed SRC and STAT3 inhibitors treatment. P Pearson correlation analysis of ICAM-1 and p-STAT3 expression levels in TCGA colon cancer patient cohort. Q The ChIP assay showed that p-STAT3 directly binds to the ICAM-1 promoters at specific sites in SW-480 cells. β-actin and positive control were used as a control for normalization. Data are presented as mean ± SD and analyzed by Student’s t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human p-CMV-ICAM-1 vector was purchased from sinobiological, pc-DNA3-SRC vector was purchased from Addgene, and p-CMV6-c-MET vector was purchased from origene.

Techniques: Kinase Assay, Expressing, Quantitative RT-PCR, Transfection, Immunohistochemistry, Staining, Transwell Chamber Assay, Tube Formation Assay, Light Microscopy, Western Blot, Real-time Polymerase Chain Reaction, Positive Control

Journal: Cell Death & Disease

Article Title: ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer

doi: 10.1038/s41419-022-04862-1

Figure Lengend Snippet: A Co-IP was performed. The cell lysate was immunoprecipitated with an ICAM-1 and SRC antibody or an immunoglobulin G (lgG) to detect the protein interaction between ICAM-1 and SRC in SW-480 and HCT-116 cells. B Representative confocal images of in situ PLA staining in SW-480 cells using anti-ICAM-1 (M) and anti-SRC (Rb). The graph shows the number of dots per cell counted using ImageJ software. Scale bar = 100 μm. C Co-IP analysis shows protein interaction of ICAM-1 with SRC using transfected HT-29 and HEK293T cells. D Co-IP assay is showed that SRC binds to the intracellular domain of ICAM-1. HEK293T cells were co-transfected with expression plasmids encoding FLAG–SRC and deletion construct of HA-ICAM-1. E Schematic diagram of the point mutation of Tyr residue in ICAM-1. F Co-IP experiment between SRC and point mutation structure of ICAM-1. HEK293T cells were co-transfected with expression plasmids encoding FLAG–SRC and point mutation construct of HA-ICAM-1. WT, wild-type ICAM-1; YA, ICAM-1 Y512A mutant (inactivation form); YD, ICAM-1 Y512D (activation form). G Representative confocal images of in situ PLA staining in HEK293T cells using anti-FLAG (M) and anti-HA (Rb). The graph shows the number of dots per cell counted using ImageJ software. Scale bar = 100 μm. H , I p-SRC activity was detected using Src kinase assay and Western blot in HT-29 cells transfected with various ICAM-1 point mutation constructs. J qRT-PCR of representative SRC signature genes was performed in HT-29 cells. K Migration/Invasion assays of ICAM-1 point mutation constructs in HT-29 cells. L qRT-PCR for marker and transcription factor of EMT were performed. M Tube formation assay of ICAM-1 point mutation constructs in HT-29 cells. Tube formation was assessed after 2 h using light microscopy, and the Image J program was used to analyze tube length. N qRT-PCR for angiogenic factors were performed. β-actin and positive control were used as a control for normalization. Data are presented as mean ± SD and analyzed by Student’s t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human p-CMV-ICAM-1 vector was purchased from sinobiological, pc-DNA3-SRC vector was purchased from Addgene, and p-CMV6-c-MET vector was purchased from origene.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, In Situ, Staining, Software, Transfection, Expressing, Construct, Mutagenesis, Activation Assay, Activity Assay, Kinase Assay, Western Blot, Quantitative RT-PCR, Migration, Marker, Tube Formation Assay, Light Microscopy, Positive Control

Journal: Cell Death & Disease

Article Title: ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer

doi: 10.1038/s41419-022-04862-1

Figure Lengend Snippet: A RTK screening with high expression in colorectal cancer compared to normal using GSE (GSE41258, GSE9348, GSE4183, GSE15960) analysis. Fold change > 2. B , C Detection of phosphorylation and expression of ICAM-1 in SW-480 cells after SU11274 treatment (2.5 μmol/L, c-MET inhibitor). D c-MET kinase assay at the protein level. ICAM-1 was purified using an HA-tagged protein purification kit and added instead of ligand. E Western blot analysis was performed in ICAM-1 silenced SW-480 cells after 24 h treatment with HGF (30 ng/μL). F Western blot showed that the intensity of p-SRC was significantly increased when c-MET and ICAM-1 were co-expressed. G SRC kinase assay also showed that the activity of p-SRC was highly increased when c-MET and ICAM-1 were co-expressed in HT-29 cells. H co-IP assay was performed. The cell lysate was immunoprecipitated with an ICAM-1 antibody or lgG to detect the protein interaction between ICAM-1 and SRC or c-MET and ICAM-1 in SW-480 cells. I After silencing the expression of ICAM-1 in SW-480 cells, co-IP experiment between SRC and c-MET was performed. J Representative confocal images of in situ PLA staining with anti-c-MET (M) and anti-SRC (Rb) in SW-480 cells with suppressed ICAM-1 expression. The graph shows the number of dots per cell counted using ImageJ software. Scale bar = 100 μm. K Immunofluorescence analysis for p-c-MET, ICAM-1, and p-SRC protein levels in HEK293T cells overexpressing c-MET, ICAM-1, and SRC (magnification, × 200, scale bar, 50 μm). L – N Transwell chamber assay, tube formation assay, and western blot were performed using HT-29 cells with overexpressed c-MET, ICAM-1 and SRC. Tube formation was assessed after 2 h using light microscopy, and the Image J program was used to analyze tube length. Data are presented as mean ± SD and analyzed by Student’s t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human p-CMV-ICAM-1 vector was purchased from sinobiological, pc-DNA3-SRC vector was purchased from Addgene, and p-CMV6-c-MET vector was purchased from origene.

Techniques: Expressing, Kinase Assay, Purification, Protein Purification, Western Blot, Activity Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, In Situ, Staining, Software, Immunofluorescence, Transwell Chamber Assay, Tube Formation Assay, Light Microscopy

Journal: Cell Death & Disease

Article Title: ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer

doi: 10.1038/s41419-022-04862-1

Figure Lengend Snippet: A Src kinase assay was detected using luminescence in SW-480 cells after 48 h treatment with ICAM-1 neutralizing antibody (2 ug/mL). B Western blot analysis was performed by treating SW-480 cells with ICAM-1 neutralizing antibody (2 ug/mL) for 24 h and then adding HGF (30 ng/μL) for 24 h. C co-IP experiment between SRC and c-MET after treatment with ICAM-1 neutralizing antibody (2 ug/mL). D , E Migration/Invasion assay and tube formation assay performed in SW-480 cells after ICAM-1 neutralizing antibody (2 ug/mL). Tube formation was assessed after 2 h using light microscopy, and the Image J program was used to analyze tube length. F SW-480 cells (3 × 10 6 ) were injected into the cecal wall of athymic BALB/c-nude (8-week-old) mice ( n = 3), and after 4 weeks ICAM-1 ab was intraperitoneally injected. And mice were sacrificed at 6 weeks. G , H Co-IP experiments and in situ PLA staining were performed in CTR-iso and ICAM-1-ab injected mouse tissues. I H&E staining images of lung tissue from mouse groups. The graph shows the number of lung metastatic lesions in each group. J – L qRT-PCR and IHC staining analysis of expression levels for EMT markers and regulators in mouse tumor tissue. M – O qRT-PCR and IHC staining aniysis of expression levels for that angiogenesis-related markers and growth in xenograft tumor tissue. P Representative immunofluorescence staining image for CD31 (red) and VEGFA (green) in mouse tumors. β-actin and positive control were used as a control for normalization. Data are presented as mean ± SD and analyzed by Student’s t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human p-CMV-ICAM-1 vector was purchased from sinobiological, pc-DNA3-SRC vector was purchased from Addgene, and p-CMV6-c-MET vector was purchased from origene.

Techniques: Kinase Assay, Western Blot, Co-Immunoprecipitation Assay, Migration, Invasion Assay, Tube Formation Assay, Light Microscopy, Injection, In Situ, Staining, Quantitative RT-PCR, Immunohistochemistry, Expressing, Immunofluorescence, Positive Control

Journal: Cell Death & Disease

Article Title: ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer

doi: 10.1038/s41419-022-04862-1

Figure Lengend Snippet: A , B Analysis of ICAM-1 expression through stagewise in CPTAC database and colorectal cancer patient tissue array. High expression of ICAM-1 was positively correlated with high-stage colorectal carcinoma. Scale bar, 100 μm. C Lymph node metastasis incidence analysis from TCGA COAD patients. D Kaplan–Meier survival analysis of lymph node positive patients according to ICAM-1 expression level from TCGA COAD patients. E Representative IHC staining images and correlation graph between ICAM-1 and p-SRC in human colorectal cancer tissue array. Percentage of groups corresponding to statistical significance according to Student’s t -test and Pearson expression correlations are indicated. Scale bar = 100 μm. F Kaplan–Meier Survival analysis of correlation between ICAM-1 and p-SRC in the colorectal cancer TCGA database. G Schematic illustration of the mechanism by which ICAM-1 may act as an adapter protein of p-SRC to regulate colorectal cancer malignancy. Data are presented as mean ± SD and analyzed by Student’s t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human p-CMV-ICAM-1 vector was purchased from sinobiological, pc-DNA3-SRC vector was purchased from Addgene, and p-CMV6-c-MET vector was purchased from origene.

Techniques: Expressing, Immunohistochemistry

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 is up-regulated by BMP2 and BMP6 treatment. (A) Microarray analysis was performed with RNA samples purified from MECs treated with BMP2, BMP6, or DMSO for 4 h . Differentially expressed genes were selected with a p-value ≤0.05 and ratio fold change ≥±1.5 and were subjected to hierarchical cluster analysis. Median-centered clusters were viewed with JavaTreeView. Fold change relative to common reference is indicated by red (+1.5; full scale) and green (−1.5; full scale) intensity. This image is a group of clustered genes with selected genes labeled. (B) MECs were treated with BMP2 and BMP6, and the total RNA was used for RT-PCR analysis with Myo10-specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (C) RT-PCR analysis of Myo10 transcripts after BMP6 treatment for the indicated times. (D) Western blotting analysis of Myo10 protein expression after BMP6 treatment for the indicated times. (E) Western blotting analysis of Myo10 protein expression after BMP6 treatment for 8 h with the indicated dosages. (F–I) Myo10 is up-regulated by BMP6 and VEGF but not by S1P and FGF-2 treatment. (F–H) MECs were treated with VEGF (F), S1P (G), and FGF2 (H) for the indicated times. The cell lysates were subjected to Western blotting analysis of Myo10 protein expression and actin as the internal control. (I) Comparison of Myo10 expression level after BMP6, VEGF, S1P, and FGF-2 treatment. The band intensity was measured and quantitated by ImageJ.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Microarray, Purification, Labeling, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Comparison

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 induced by BMP6 is localized in filopodia. MECs were treated with BMP6 for 8 h (A–C and G–I) or DMSO (D–F) and were fixed with PFA. Cells were stained with phalloidin (A, D, G, J, and M) and chicken anti–mouse Myo10 pAb (E and H) or control IgG (B) as a negative control. Exogenous Myo10 (GFP tagged) was expressed by transiently transfecting MECs with a plasmid encoding GFP-Myo10 (J–L). GFP control plasmids (M–O) were transfected in MECs as the negative controls for GFP-Myo10. Actin was visualized using Texas red–phalloidin (red). Myo10 was visualized using chicken anti-Myo10 pAb (green; E and H) or GFP (green; K and N). The arrows represent filopodia.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Staining, Control, Negative Control, Plasmid Preparation, Transfection

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 siRNAs inhibit BMP6-induced filopodia. (A) MECs transfected with Myo10 siRNAs were lysed and subjected to Western blotting analysis with anti-Myo10 pAb (top). A β-actin blot of the same membrane controlled for sample loadings (bottom). (B–H) Knockdown of endogenous Myo10 in MECs by Myo10 siRNAs decreased filopodial number in the BMP6-treated conditions. MECs were transfected with GFP-tagged Myo10 siRNA1 (C and F) or siRNA2 (D and G) or by the GFP-tagged control vector containing irrelevant siRNA sequence (B and E). Cells were treated with 100 ng/ml BMP6 (E–G) or DMSO (B–D) as a control, and GFP-positive cells were subjected for SEM analysis. (H) The bar graphs show quantification of filopodial numbers in MECs transfected with GFP-tagged control siRNA, GFP-Myo10 siRNA1, or GFP-Myo10 siRNA2. *, P < 0.05; n = 3. n = 20 cells per sample.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Transfection, Western Blot, Membrane, Knockdown, Control, Plasmid Preparation, Sequencing

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Cell alignment is dependent on Myo10. (A–D) MECs aligned toward the BMP6 gradient. Cells cultured on the coverslips were pretreated with BMP6 (B and D) or DMSO (A and C) for 4 h, were transferred to the Dunn chamber with the media containing 0–200 ng/ml BMP6 gradient (C and D) or not containing gradient (A and B), and were maintained for 4 h. Cells were fixed and stained with phalloidin, DAPI, and GM130. Actin (Texas red–phalloidin), red; Golgi (anti-GM130 mAb), green; nucleus (DAPI), blue. (E–K) Knockdown of endogenous Myo10 in MECs by Myo10 siRNAs inhibited cell alignment. (E–J) Cells were transfected with GFP-Myo10 siRNA1 or GFP-Myo10 siRNA2 and controlled by transfecting cells with GFP-tagged control vector containing irrelevant siRNA sequence. The cells were pretreated with BMP6 for 4 h and were transferred into the Dunn chamber for another 4 h with 0–200 ng/ml of the media containing BMP6 gradient. The cells were fixed, and immunofluorescent images were taken. GFP, green; Golgi (anti-GM130 mAb), red; nucleus (DAPI), blue. White arrowheads represent the transfected cells with siRNAs (GFP positive). (K) Quantitative analysis of the aligned cells toward the higher BMP6 gradient. Error bars represent SD. n = 3; *, P < 0.05. n = at least 57 cells per sample. p.t., pretreatment.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Cell Culture, Staining, Knockdown, Transfection, Control, Plasmid Preparation, Sequencing

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 knockdown inhibits directed cell migration and angiogenesis induced by BMP6. (A–E) BMP6 increased Myo10 proteins in filopodia, filopodial number, and directed migration. Wound-healing assay was performed with MECs grown on 35-mm wells. (A) The bar graph shows the recovered area after cells were stimulated with BMP6 for 16 h. (B and D) The immunofluorescent staining of cells. Actin (Texas red–phalloidin), red; Myo10 (anti-Myo10 pAb), green; overlay of the red and green, yellow. The increased yellow staining demonstrated the increased localization of Myo10 protein at the filopodial tips of the cells on the leading edge, where the filopodial number also increased. (C and E) The increase of filopodial number in SEM images of cells. Arrows represent the migration direction of the cells. IF, immunofluorescence; SEM, scanning EM. (F) Myo10 knockdown inhibited directed cell migration induced by BMP6. MECs were transfected with GFP-Myo10 siRNA1, GFP-Myo10 siRNA2, or control siRNA, were pretreated with BMP6 for 4 h, and were trypsinized for the Boyden chamber assay. The cells migrating through the filter were counted. p.t., pretreatment. (G–M) Myo10 knockdown inhibited tube formation induced by BMP6 using 3D collagen assay. MECs were transfected with GFP-Myo10 siRNA1, GFP-Myo10 siRNA2, or control siRNA and were lysed for the 3D collagen angiogenesis assay. (G–L) The brightfield images were taken after 24 h of incubation. (M) Quantitation of the tube formation. Error bars represent SD. n = 3; *, P < 0.05.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Knockdown, Migration, Wound Healing Assay, Staining, Immunofluorescence, Transfection, Control, Boyden Chamber Assay, Collagen Assay, Angiogenesis Assay, Incubation, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 interacts with ALK6. (A) Selected sequence from a time-lapse video focusing on the movements of puncta containing GFP-ALK6 proteins. Arrows represent the puncta containing GFP-ALK6. The images were taken every 30 s for 120 s after MECs were treated with BMP6 for 4 h. The mean rate of the forward movements for the puncta was ∼42 ± 16 nm s −1 ( n = 10 puncta). (B) Colocalization of exogenous GFP-tagged Myo10 and HA-tagged ALK6 at tips of filopodia in HeLa cells. Myo10 (GFP), green; ALK6 (anti-HA mAb), red. The arrow indicates colocalization. (C) Exogenous Myo10 associated with exogenous ALK6. GFP-Myo10 and HA-ALK6 were expressed in 293T cells, and immunoprecipitation was performed with anti-HA mAb followed by Western blotting against GFP. (D) Endogenous Myo10 associated with ALK6. MEC lysates were immunoprecipitated by anti-ALK6 mAb, with mouse IgG as a control, followed by Western blotting against Myo10.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Sequencing, Immunoprecipitation, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 knockdown inhibits BMP6-induced Smad1, 5, and 8 activation. MECs were transfected with Myo10 siRNA2 or control siRNA and were stimulated with BMP6 for the indicated times for the activation of Smads. Cells were lysed for Western blotting analysis of the antiphospho-Smad1, 5, and 8 pAb, anti-Smad5 pAb, and anti-Myo10 pAb. This is a representative image of three independent experiments.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Knockdown, Activation Assay, Transfection, Control, Western Blot

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Schematic model for Myo10 as a sensor for filopodia to sense the BMP6 gradient for directed cell migration and angiogenesis.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Migration

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: DNA sequences of the primers used for traditional and real time RT-PCR

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Amplification

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Microarray, Expressing, Sequencing, Ubiquitin Proteomics, Binding Assay, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Induction of chemokine gene expression by LCN2 in astrocytes, microglia, endothelial cells, and neuron cells. Astrocytes (A and E), microglia (B and E), bEnd.3 endothelial cells (C), and neuron cells (D) were treated with the recombinant LCN2 protein (10 μg/ml) for 8 h, and the total RNA was isolated for traditional RT-PCR or real time PCR. The cells were also treated for 8 h with LPS (100 ng/ml), TNF-α (10 ng/ml), or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for comparison purposes. The mRNA levels of chemokines (CCL4, CCL20, CXCL2, and CXCL10) and other inflammatory genes (IL-6, COX-2, iNOS, and PIAS3) were determined by traditional RT-PCR (A–D) or real time PCR (E). β-Actin or GAPDH was used as an internal control. The results are one representative of more than three independent experiments (A–D) or means ± S.D. (n = 3) (E).

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Gene Expression, Recombinant, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: An increase of CXCL10 expression by LCN2 in astrocytes. Astrocytes were incubated with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h. The amounts of CXCL10 protein in the culture media were measured by specific ELISA (A). The results are means ± S.D. (n = 3). *, p < 0.05 compared with the untreated control. The stable overexpression or knockdown of lcn2 expression was achieved by transfection with sense or antisense lcn2 cDNA in C6 rat glioma cells. The increased or decreased lcn2 expression in the stable transfectants (S3, lcn2 sense transfectant; AS7, lcn2 antisense transfectant) compared with cells transfected with an empty vector (V2) was confirmed by RT-PCR (B). Changes in the CXCL10 mRNA levels in the stable transfectants were also assessed by RT-PCR (C). β-Actin was used as an internal control. The results are one representative of more than three independent experiments.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Expressing, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Astrocyte-derived CXCL10 promoted the migration of astrocytes. Astrocytes (1 × 104 cells/upper well) were exposed to LCN2 (10 μg/ml)-stimulated ACM or the recombinant CXCL10 protein (10 ng/ml) in the presence or absence of CXCL10 neutralizing antibody (CXCL10 Ab; 10 ng/ml) as indicated. ACM-None, untreated ACM; ACM-LCN2, LCN2-treated ACM (see “Experimental Procedures” for the preparation of ACM). After treatment for the indicated time periods, either wound healing assay (A) or the Boyden chamber assay (B) was performed to evaluate cell migration. A representative microscopic image for each condition was shown (magnification, ×100) (upper). The quantification of cell migration was done by either measuring the degree of wound closure (wound healing assay) or enumerating the migrated cells (Boyden chamber assay) as described under “Experimental Procedures” (lower). The results are means ± S.D. (n = 3). *, p < 0.05 compared with ACM-None at the same time point; **, p < 0.05 compared with ACM-LCN2 at the same time point; #, p < 0.05 compared with the untreated control (None) at the same time point. For the checkerboard analysis, migration of astrocytes (2 × 104 cells/upper well) in response to the indicated concentrations of ACM-LCN2 (C) and the recombinant CXCL10 protein (D) placed in upper and/or lower well was determined using the Boyden chamber assay. The quantification of cell migration was done by enumerating the migrated cells after 48 h as described under “Experimental Procedures.” The results are the means ± S.D. (n = 3). *, p < 0.05 between the treatments indicated.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Derivative Assay, Migration, Recombinant, Wound Healing Assay, Boyden Chamber Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Checkerboard analysis for promigratory effects of recombinant CXCL10 protein in astrocytes The recombinant CXCL10 protein was added to the upper and/or lower wells of the Boyden chambers for the checkerboard analysis. After cells were incubated at 37 °C under 5% CO 2 for 48 h, cell migration was assessed as described under “Experimental Procedures.” NT, not tested.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Recombinant, Incubation, Migration

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: The effects of LCN2 protein expressed in mammalian cells (mLCN2) on the chemokine gene expression and cell migration in astrocytes. Astrocytes were treated with the NSO murine melanoma cell-derived mouse LCN2 protein (10 μg/ml; mLCN2) for 8 h, and the total RNA was isolated for traditional RT-PCR. The cells were also treated for 8 h with LPS (100 ng/ml) plus IFN-γ (50 units/ml) for comparison purposes. The mRNA levels of CXCL10 and PIAS3 were determined by traditional RT-PCR (A). β-Actin was used as an internal control. The results are one representative of more than three independent experiments. Astrocytes (1 × 104 cells/upper well) were exposed to the melanoma cell-expressed LCN2 protein (10 μg/ml; mLCN2)-stimulated astrocyte-conditioned media (ACM-mLCN2). Astrocytes placed in the Boyden chambers were then incubated at 37 °C for 24–72 h to evaluate cell migration (B). A representative microscopic image for each condition was shown (magnification, ×100) (upper). ACM-None, untreated ACM; ACM-mLCN2, mLCN2-treated ACM. The quantification of cell migration was done by enumerating the migrated cells as described under “Experimental Procedures” (lower). The results are the means ± S.D. (n = 3). *, p < 0.05 compared with ACM-None at the same time point.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Gene Expression, Migration, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Comparison, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Astrocyte-derived CXCL10 promoted the migration of microglia and neuron cells. Microglia (1 × 104 cells/upper well) (A) or neuron cells (1 × 104 cells/upper well) (B) were exposed to LCN2 (10 μg/ml)-stimulated ACM or the recombinant CXCL10 protein (10 ng/ml) in the presence or absence of CXCL10 neutralizing antibody (10 ng/ml) as indicated. Microglia or neuron cells placed in the Boyden chambers were incubated at 37 °C for 12–48 or 18–48 h, respectively, to evaluate cell migration. The GST protein (10 μg/ml) was used as a control for the recombinant LCN2 protein. A representative microscopic image for each condition is shown (magnification, ×100) (upper). ACM-None, untreated ACM; ACM-LCN2, LCN2-treated ACM. The quantification of cell migration was done by enumerating the migrated cells as described under “Experimental Procedures” (lower). The results are the means ± S.D. (n = 3). *, p < 0.05 compared with ACM-None at the same time point; **, p < 0.05 compared with ACM-LCN2 at the same time point; #, p < 0.05 compared with the untreated control (None) at the same time point.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Derivative Assay, Migration, Recombinant, Incubation, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: JAK2/STAT3 and IKK/NF-κB mediated LCN2 up-regulation of CXCL10 and GFAP expression in astrocytes. Astrocytes were pretreated with the recombinant LCN2 protein (10 μg/ml) for 24 h prior to the treatment with phorbol 12-myristate 13-acetate (100 μg/ml), ATP (3 mm), or IFN-γ (50 units/ml) for 30 min. Astrocytes were also exposed to phorbol 12-myristate 13-acetate (PMA), ATP, or IFN-γ for 30 min without LCN2 pretreatment (A). Alternatively, astrocytes were treated with LCN2 for 0.5–24 h for the time kinetics analysis (B). The levels of phosphorylated STAT3 (pSTAT3 at Ser727 or Tyr705) or total STAT3 protein were then evaluated by Western blot analysis. The results are one representative of more than three independent experiments. Alternatively, astrocytes were pretreated with AG490 (JAK2/STAT3-specific inhibitor, 50 μm) or piceatannol (JAK1/STAT1-specific inhibitor, 50 μm) for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h. The secreted CXCL10 protein was measured by specific ELISA (C). The results are the means ± S.D. (n = 3). *, p < 0.001 compared with the treatment without inhibitors. Astrocytes were pretreated with AG490 for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) for 24 h. The expression of GFAP protein levels was assessed by Western blot analysis, respectively (D). After astrocytes were treated with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 1 h, an EMSA analysis of the nuclear extracts was conducted by using a 32P-labeled NF-κB oligonucleotide probe (E). Binding specificity was determined by the supershift assay using antibody against p65 (p65 Ab) or its coincubation with an unlabeled probe containing the NF-κB binding sequence (cold probe) to compete with the labeled oligonucleotide. The results are one representative of more than three independent experiments. Primary astrocytes were pretreated with pyrrolidine dithiocarbamate (PTDC, NF-κB-specific inhibitor; 0–10 μm) for 30 min prior to their treatment with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h (F). The concentration of nitrite in the culture media was measured by the Griess reagent. The results are the means ± S.D. (n = 3). *, p < 0.001 compared with the LCN2 or LPS/IFN-γ treatment alone. The astrocytes were pretreated with SC-514 (IKK-specific inhibitor, 10 μm) for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) for 8–24 h. The expression of GFAP at mRNA or protein levels after 8 or 24 h was then assessed by RT-PCR or Western blot analysis, respectively (G).

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Expressing, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, Sequencing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Role of LCN2 in astrocyte migration and CXCL10 induction in cortical stab wound injury model. Cortical stab wound injury was performed with LCN2 wild-type (LCN2+/+) or LCN2-deficient mice (LCN2−/−) (A, upper). At 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against GFAP. The asterisk indicates stab wound injury site. The boxes indicate 200-μm × 200-μm squares placed for cell counting. Immunohistochemistry results showed that GFAP-positive cells in the peri-injury region were observed in both LCN2+/+ and LCN2−/− mice. A significant decrease in the number of GFAP-positive cells was observed in the immediate vicinity of injury site in LCN2−/− mice (A, lower panel). The results are one representative of more than three independent experiments. Scale bars, 200 μm. The values are the means ± S.D. from three different animals and six independent sections/animal. *, p < 0.05 compared with LCN2−/− mice in the same counting area; #, p < 0.05 between the values indicated. The mRNA levels of lcn2 (upper panel) and CXCL10 (lower panel) in LCN2+/+ and LCN2−/− mice were examined at 2 days after cortical stab wound injury (B). RNA was isolated from the injury site in the cortex and subjected to real time PCR. The injury-induced CXCL10 expression was completely abrogated in LCN2−/− brain as compared with LCN2+/+ littermates. GAPDH was used as a control in the real time PCR. The results are the means ± S.D. (n = 3). *, p < 0.05 compared with uninjured LCN2+/+ mice; **, p < 0.05 compared with injured LCN2+/+ mice; #, p < 0.05 compared with uninjured LCN2−/− mice.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Migration, Cell Counting, Immunohistochemistry, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Essential role of LCN2 in reactive astrocytosis and CXCL10 induction in LPS-induced mouse neuroinflammation models. LCN2+/+ or LCN2−/− were injected with LPS intracortically (A) or icv (B). After 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against LCN2 (green, upper) or GFAP (red, middle). The nuclei were counterstained with DAPI (blue, lower). The asterisk indicates the injection site. A significant decrease in both LCN2 and GFAP expression was observed in LCN2−/− mice. The results are one representative of more than three independent experiments. Scale bars, 100 μm. Quantification of the GFAP-positive cells was done in the cortex (A, upper graph) or hippocampus (B, upper graph). The values are the means ± S.D. from three different animals and five independent sections per animal. The mRNA levels of lcn2, CXCL10, and GFAP in LCN2+/+ and LCN2−/− mice were examined by real time PCR of cortical tissue around the injection site (A, lower graph) or hippocampus (B, lower graph) at 2 days after intracortical or icv injection with LPS, respectively. LCN2−/− mice exhibited markedly lower levels of CXCL10 and GFAP as compared with LCN2+/+ littermates. *, p < 0.05; compared with wild-type LPS-injected mice (LCN2+/+) at the same inflammation model. At 2 days after icv injection of LCN2+/+ mice with LPS, hippocampus was immunostained with antibodies against LCN2 (green) or GFAP (red) (C). The nuclei were counterstained with DAPI (blue). A merged image is shown in the lower right panel. The arrowheads indicate colocalization of LCN2 and GFAP (yellow). Cell bodies and processes of astrocytes in hippocampus were stained with LCN2 antibody. The results are representative of more than three independent experiments. Scale bars, 20 μm.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Schematic diagram depicting the promotion of CNS cell migration by LCN2-induced CXCL10 (A) and the possible pathway through which LCN2 induces astrocyte migration and morphological changes (B). A, LCN2 up-regulates CXCL10 expression in the multiple cell types in the CNS, such as astrocytes, microglia, neurons, and endothelial cells. Astrocyte-derived CXCL10 acts in a paracrine or autocrine manner to promote cell migration in the inflammatory scene. CXCL10 secreted by other cell types may play a similar role. The lcn2 receptor and CXCL10 receptor (CXCR3) are widely expressed in glia, endothelial cells, and neurons. B, LCN2 up-regulates CXCL10 and GFAP expression in reactive astrocytes through JAK2/STAT3 and NF-κB pathways. Although LCN2-up-regulated CXCL10 promotes cell migration, GFAP induction may lead to morphological changes observed in reactive astrocytosis. Based on the microarray analysis, LCN2 induces the up-regulation of IL-6 and down-regulation of PIAS3, thereby facilitating the STAT3 pathway (dotted line). NO production, downstream of the NF-κB, may cooperate with the STAT3 pathway to induce GFAP expression. NO has been shown to induce GFAP expression in astrocytes (bold dotted line) (54). Moreover, IL-6 previously induced GFAP expression through the STAT3 pathway (96). Other pathways may also participate in astrocyte migration and morphological change under the current conditions.

Article Snippet: The recombinant mouse CXCL10 protein (R & D Systems) was used as a standard.

Techniques: Migration, Expressing, Derivative Assay, Microarray

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 1. Induction of chemokine gene expression by LCN2 in astrocytes, microglia, endothelial cells, and neuron cells. Astrocytes (A and E), microglia (B and E), bEnd.3 endothelial cells (C), and neuron cells (D) were treated with the recombinant LCN2 protein (10 g/ml) for 8 h, and the total RNA was isolated for traditional RT-PCR or real time PCR. The cells were also treated for 8 h with LPS (100 ng/ml), TNF- (10 ng/ml), or LPS (100 ng/ml) plus IFN- (50 units/ml) for comparison purposes. The mRNA levels of chemokines (CCL4, CCL20, CXCL2, and CXCL10) and other inflammatory genes (IL-6, COX-2, iNOS, and PIAS3) were determined by traditional RT-PCR (A–D) or real time PCR (E). -Actin or GAPDH was used as an internal control. The results are one representative of more than three independent experiments (A–D) or means S.D. (n 3) (E).

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Gene Expression, Recombinant, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 2. An increase of CXCL10 expression by LCN2 in astrocytes. Astro- cytes were incubated with the recombinant LCN2 protein (10 g/ml) or LPS (100 ng/ml) plus IFN- (50 units/ml) for 24 h. The amounts of CXCL10 protein in the culture media were measured by specific ELISA (A). The results are means S.D. (n 3). *, p 0.05 compared with the untreated control. The stable overexpression or knockdown of lcn2 expression was achieved by transfection with sense or antisense lcn2 cDNA in C6 rat glioma cells. The increased or decreased lcn2 expression in the stable transfectants (S3, lcn2 sense transfectant; AS7, lcn2 antisense transfectant) compared with cells transfected with an empty vector (V2) was confirmed by RT-PCR (B). Changes in the CXCL10 mRNA levels in the stable transfectants were also assessed by RT-PCR (C). -Actin was used as an internal control. The results are one repre- sentative of more than three independent experiments.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Expressing, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 3. Astrocyte-derived CXCL10 promoted the migration of astrocytes. Astrocytes (1 104 cells/upper well) were exposed to LCN2 (10 g/ml)- stimulated ACM or the recombinant CXCL10 protein (10 ng/ml) in the presence or absence of CXCL10 neutralizing antibody (CXCL10 Ab; 10 ng/ml) as indicated. ACM-None, untreated ACM; ACM-LCN2, LCN2-treated ACM (see “Experimental Procedures” for the preparation of ACM). After treatment for the indicatedtimeperiods,eitherwoundhealingassay(A)ortheBoydenchamberassay(B)wasperformedtoevaluatecellmigration.Arepresentativemicroscopic image for each condition was shown (magnification, 100) (upper). The quantification of cell migration was done by either measuring the degree of wound closure (wound healing assay) or enumerating the migrated cells (Boyden chamber assay) as described under “Experimental Procedures” (lower). The results are means S.D. (n 3). *, p 0.05 compared with ACM-None at the same time point; **, p 0.05 compared with ACM-LCN2 at the same time point; #, p 0.05 compared with the untreated control (None) at the same time point. For the checkerboard analysis, migration of astrocytes (2 104 cells/upper well) in response to the indicated concentrations of ACM-LCN2 (C) and the recombinant CXCL10 protein (D) placed in upper and/or lower well was determined using the Boyden chamber assay. The quantification of cell migration was done by enumerating the migrated cells after 48 h as described under “Experimental Procedures.” The results are the means S.D. (n 3). *, p 0.05 between the treatments indicated.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Derivative Assay, Migration, Recombinant, Wound Healing Assay, Boyden Chamber Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 4. The effects of LCN2 protein expressed in mammalian cells (mLCN2) on the chemokine gene expression and cell migration in astrocytes. Astrocytes were treated with the NSO murine melanoma cell- derived mouse LCN2 protein (10 g/ml; mLCN2) for 8 h, and the total RNA was isolated for traditional RT-PCR. The cells were also treated for 8 h with LPS (100 ng/ml) plus IFN- (50 units/ml) for comparison purposes. The mRNA levels of CXCL10 and PIAS3 were determined by traditional RT-PCR (A). -Actin was used as an internal control. The results are one represen- tative of more than three independent experiments. Astrocytes (1 104 cells/upper well) were exposed to the melanoma cell-expressed LCN2 pro- tein (10 g/ml; mLCN2)-stimulated astrocyte-conditioned media (ACM- mLCN2). Astrocytes placed in the Boyden chambers were then incubated at 37 °C for 24–72 h to evaluate cell migration (B). A representative micro- scopic image for each condition was shown (magnification, 100) (upper). ACM-None, untreated ACM; ACM-mLCN2, mLCN2-treated ACM. The quan- tification of cell migration was done by enumerating the migrated cells as described under “Experimental Procedures” (lower). The results are the means S.D. (n 3). *, p 0.05 compared with ACM-None at the same time point.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Gene Expression, Migration, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Comparison, Control, Incubation

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 5. Astrocyte-derived CXCL10 promoted the migration of microglia and neuron cells. Microglia (1 104 cells/upper well) (A) or neuron cells (1 104 cells/upper well) (B) were exposed to LCN2 (10 g/ml)-stimulated ACM or the recombinant CXCL10 protein (10 ng/ml) in the presence or absence of CXCL10 neutralizing antibody (10 ng/ml) as indicated. Microglia or neuron cells placed in the Boyden chambers were incubated at 37 °C for 12–48 or 18–48 h, respectively, to evaluate cell migration. The GST protein (10 g/ml) was used as a control for the recombinant LCN2 protein. A representative microscopic image for each condition is shown (magnification, 100) (upper). ACM-None, untreated ACM; ACM-LCN2, LCN2-treated ACM. The quantification of cell migration was done by enumerating the migrated cells as described under “Experimental Procedures” (lower). The results are the means S.D. (n 3). *, p 0.05 compared with ACM-None at the same time point; **, p 0.05 compared with ACM-LCN2 at the same time point; #, p 0.05 compared with the untreated control (None) at the same time point.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Derivative Assay, Migration, Recombinant, Incubation, Control

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 6. The effect of LCN2-treated ACM on the morphology of astro- cytesandmicroglia.ACMwaspreparedafterthetreatmentofprimaryastro- cytes with LCN2 (10 g/ml) for 24 h. The addition of LCN2-treated ACM (ACM- LCN2) induced morphological changes in primary astrocytes and primary microglia cultures after 24 h (A). Primary astrocytes were stained with GFAP antibody (original magnification, 400), followed by the incubation with anti-mouse IgG-FITC-conjugated secondary antibody. Primary microglia were stained with the peroxidase-labeled isolectin B4 (original magnifica- tion, 100), followed by incubation with diaminobenzidine tetrahydrochlo- ride. Scale bars, 25 m. The length of the longest process in each astrocyte or the percentage of ramified microglia was assessed by examining several ran- domly chosen microscopic fields (B). The results are the means S.D. (n 3). *, p 0.05; compared with the untreated ACM control (ACM-None).

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Staining, Incubation, Labeling, Control

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 7. JAK2/STAT3 and IKK/NF-B mediated LCN2 up-regulation of CXCL10 and GFAP expression in astrocytes. Astrocytes were pretreated with the recombinant LCN2 protein (10 g/ml) for 24 h prior to the treatment with phorbol 12-myristate 13-acetate (100 g/ml), ATP (3 mM), or IFN- (50 units/ml) for 30 min. Astrocytes were also exposed to phorbol 12-myristate 13-acetate (PMA), ATP, or IFN- for 30 min without LCN2 pretreatment (A). Alternatively, astrocytes were treated with LCN2 for 0.5–24 h for the time kinetics analysis (B). The levels of phosphorylated STAT3 (pSTAT3 at Ser727 or Tyr705) or total STAT3 protein were then evaluated by Western blot analysis. The results are one representative of more than three independent experiments. Alternatively, astro- cyteswerepretreatedwithAG490(JAK2/STAT3-specificinhibitor,50M)orpiceatannol(JAK1/STAT1-specificinhibitor,50M)for30minpriortothetreatment withtherecombinantLCN2protein(10g/ml)orLPS(100ng/ml)plusIFN-(50units/ml)for24h.ThesecretedCXCL10proteinwasmeasuredbyspecificELISA (C). The results are the means S.D. (n 3). *, p 0.001 compared with the treatment without inhibitors. Astrocytes were pretreated with AG490 for 30 min prior to the treatment with the recombinant LCN2 protein (10 g/ml) for 24 h. The expression of GFAP protein levels was assessed by Western blot analysis, respectively (D). After astrocytes were treated with the recombinant LCN2 protein (10 g/ml) or LPS (100 ng/ml) plus IFN- (50 units/ml) for 1 h, an EMSA analysis of the nuclear extracts was conducted by using a 32P-labeled NF-B oligonucleotide probe (E). Binding specificity was determined by the supershift assay using antibody against p65 (p65 Ab) or its coincubation with an unlabeled probe containing the NF-B binding sequence (cold probe) to compete with the labeled oligonucleotide. The results are one representative of more than three independent experiments. Primary astrocytes were pretreated with pyrrolidine dithiocarbamate (PTDC, NF-B-specific inhibitor; 0–10 M) for 30 min prior to their treatment with the recombinant LCN2 protein (10 g/ml) or LPS (100 ng/ml) plus IFN- (50 units/ml) for 24 h (F). The concentration of nitrite in the culture media was measured by the Griess reagent. The results are the means S.D. (n 3). *, p 0.001 compared with the LCN2 or LPS/IFN- treatment alone. The astrocytes were pretreated with SC-514 (IKK-specific inhibitor, 10 M) for 30 min prior to the treatment with the recombinant LCN2 protein (10 g/ml) for 8–24 h. The expression of GFAP at mRNA or protein levels after 8 or 24 h was then assessed by RT-PCR or Western blot analysis, respectively (G).

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Expressing, Recombinant, Western Blot, Labeling, Binding Assay, Sequencing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 8. Role of LCN2 in astrocyte migration and CXCL10 induction in cortical stab wound injury model. Cortical stab wound injury was performed with LCN2 wild-type (LCN2/) or LCN2-deficient mice (LCN2/) (A, upper). At 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against GFAP. The asterisk indicates stab wound injury site. The boxes indicate 200-m 200-m squares placed for cell counting. Immunohisto- chemistry results showed that GFAP-positive cells in the peri-injury region were observed in both LCN2/ and LCN2/ mice. A significant decrease in the number of GFAP-positive cells was observed in the immediate vicinity of injury site in LCN2/ mice (A, lower panel). The results are one representative of more than three independent experiments. Scale bars, 200 m. The values are the means S.D. from three different animals and six independent sections/animal. *,p 0.05comparedwithLCN2/miceinthesamecountingarea;#,p 0.05betweenthevaluesindicated.ThemRNAlevelsoflcn2(upperpanel)andCXCL10 (lower panel) in LCN2/ and LCN2/ mice were examined at 2 days after cortical stab wound injury (B). RNA was isolated from the injury site in the cortex and subjected to real time PCR. The injury-induced CXCL10 expression was completely abrogated in LCN2/ brain as compared with LCN2/ littermates. GAPDH was used as a control in the real time PCR. The results are the means S.D. (n 3). *, p 0.05 compared with uninjured LCN2/ mice; **, p 0.05 compared with injured LCN2/ mice; #, p 0.05 compared with uninjured LCN2/ mice.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Migration, Cell Counting, Immunohistochemistry, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE9.EssentialroleofLCN2inreactiveastrocytosisandCXCL10inductioninLPS-inducedmouseneuroinflammationmodels.LCN2/orLCN2/ were injected with LPS intracortically (A) or icv (B). After 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against LCN2 (green, upper) or GFAP (red, middle). The nuclei were counterstained with DAPI (blue, lower). The asterisk indicates the injection site. A significant decrease in both LCN2 and GFAP expression was observed in LCN2/ mice. The results are one representative of more than three independent experiments. Scale bars, 100m.QuantificationoftheGFAP-positivecellswasdoneinthecortex(A,uppergraph)orhippocampus(B,uppergraph).ThevaluesarethemeansS.D.from three different animals and five independent sections per animal. The mRNA levels of lcn2, CXCL10, and GFAP in LCN2/ and LCN2/ mice were examined by real time PCR of cortical tissue around the injection site (A, lower graph) or hippocampus (B, lower graph) at 2 days after intracortical or icv injection with LPS, respectively. LCN2/ mice exhibited markedly lower levels of CXCL10 and GFAP as compared with LCN2/ littermates. *, p 0.05; compared with wild-type LPS-injected mice (LCN2/) at the same inflammation model. At 2 days after icv injection of LCN2/ mice with LPS, hippocampus was immunostained with antibodies against LCN2 (green) or GFAP (red) (C). The nuclei were counterstained with DAPI (blue). A merged image is shown in the lower right panel. The arrowheads indicate colocalization of LCN2 and GFAP (yellow). Cell bodies and processes of astrocytes in hippocampus were stained with LCN2 antibody. The results are representative of more than three independent experiments. Scale bars, 20 m.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.m111.299248

Figure Lengend Snippet: FIGURE 10. Schematic diagram depicting the promotion of CNS cell migration by LCN2-induced CXCL10 (A) and the possible pathway through which LCN2 induces astrocyte migration and morphological changes (B). A, LCN2 up-regulates CXCL10 expression in the multiple cell types in the CNS, such as astrocytes, microglia, neurons, and endothelial cells. Astrocyte-derived CXCL10 acts in a paracrine or autocrine manner to promote cell migration in the inflammatory scene. CXCL10 secreted by other cell types may play a similar role. The lcn2 receptor and CXCL10 receptor (CXCR3) are widely expressed in glia, endothelial cells, and neurons. B, LCN2 up-regulates CXCL10 and GFAP expression in reactive astrocytes through JAK2/STAT3 and NF-B pathways. Although LCN2-up-regulated CXCL10 promotes cell migration, GFAP induction may lead to morphological changes observed in reactive astrocytosis. Based on the microarray analysis, LCN2 induces the up-regulation of IL-6 and down-regulation of PIAS3, thereby facilitating the STAT3 pathway (dotted line). NO production, downstream of the NF-B, may cooperate with the STAT3 pathway to induce GFAP expression. NO has been shown to induce GFAP expression in astrocytes (bold dotted line) (54). Moreover, IL-6 previously induced GFAP expression through the STAT3 pathway (96). Other pathways may also participate in astrocyte migration and morphological change under the current conditions.

Article Snippet: The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit polyclonal anti-phospho-STAT3 at Ser727/Tyr705 and anti-total STAT3 antibodies (Cell Signaling Technology, Beverly,MA);mousemonoclonal anti-GFAP antibody (Biogenex); goat polyclonal anti-mouse LCN2 antibody (R & D Systems); and monoclonal anti- -tubulin clone B-5– TABLE 2 Partial list of genes that were up or down-regulated by LCN2 as determined by DNA microarray analysis of astrocytes Genes whose expression was increased or decreased greater than 1.5- or 3.5-fold, respectively, by LCN2 were listed.

Techniques: Migration, Expressing, Derivative Assay, Microarray

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). (B) Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including non-phospho-TM peptide during the antibody incubation reduces the detection of non-phosphorylated PKN. (C) IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. (B) The PKN1 TM mutant S916A has reduced kinase activity towards multiple substrates. (C) Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. (D, E) The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 hrs, and analyzed by using pan- and phosphosite-specific antibodies. (B) Cells were treated with torin and rapamycin during a time course up to 24 hrs and subsequently analyzed by using pan- and phosphosite-specific antibodies.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. (B) Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. (C) Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. (D) Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. (E) Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (**** p =< 0.0001). (G) Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. (B) PKN1 and PKN2 expression (using microarray data from [47]) in normal prostate, primary tumor, and metastases. (C) RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT and select mTOR components. ** p =< 0.01 *** p =< 0.001 **** p =< 0.0001

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of weaned mice.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of weaned mice from double heterozygous intercrosses.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β-galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2mm, 0.5mm, 1.0mm. B) Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75 and E9.5. Scale bars 0.2mm (upper four panels), 1.0mm. (C) Whole mount images of wild type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2mm.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). (B–E) H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1; Tg-PKN1, 41 weeks; Tg-AKT1; Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP; Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP; Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Fig. 3).

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Prostate phenotypes in the ventral lobe

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200x magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r ;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r ;Pkn2r/r, 26 and 45 weeks. (D) The images of invasive cancer (left and middle) are from 53 week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53 week animal. Lower magnification views of the same samples are also provided (Supplemental Fig. 4).

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Staining