Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: (A) Spleen cells from cotton rats were stimulated with Concanavalin A for 72 hours and lymphoblasts purified by centrifugation on a ficoll-gradient. Subsequently, cells were stained with antisera specific for the human interferon receptor to test for crossreactive binding to cotton rat cells and analyzed by flow cytometry. Antibody specific for human interferon alpha receptor-1 (IFNAR-1) did not react with cotton rat cells (left) whereas antibody specific for human interferon alpha receptor-2 (IFNAR-2) did (right). IFNR specific antibody is shown as black line, controls as gray areas. B. IFNAR-2 antibody was added to cotton rat spleen cells. One hour later different concentrations of a combination of ODN2216 and poly I:C was added. 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments. Significant differences were seen between cells treated with the combination of ODN2216 and poly I:C alone, or the combination of ODN2216 and poly I:C with antibody. IFNAR-1 antibody (not reactive with cotton rat cells) had no effect. C. Cotton rat spleen cells were stimulated with ODN 2216 or poly I:C, with or without the addition of recombinant cotton rat interferon alpha. (One unit equals one picogram of type I interferon.) 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments, and significance was compared to cells without type I interferon treatment. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Purification, Centrifugation, Staining, Binding Assay, Flow Cytometry, Recombinant

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: A) The number of MeV-specific B cells was measured from bone marrow cells of MeV-immune cotton rats. The addition of ODN 2216 and poly I:C individually and in combination increased B cell numbers whereas the addition of sera neutralizing cotton rat interferon alpha and IL-6 reduced this stimulation. Each bar graph represents the mean ± SD of triplicate wells. B) Cotton rats were immunized intranasally (upper panel) or subcutaneously (lower panel) with MeV, or MeV with ODN 2216 and/or pI:C in the presence or absence of human MeV-specific IgG (neutralization titer of 100) which had been injected intraperitoneally one day before immunization. Sera were collected at seven weeks post vaccination and the titer of neutralizing antibody was determined by neutralization assay. Each bar graph represents the average titer of four animals ± SD. The experiment is representative of three experiments. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Neutralization, Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Clinical significance of PD-L1 + exosomes in plasma of Head and Neck Cancer patients

doi: 10.1158/1078-0432.CCR-17-2664

Figure Lengend Snippet: Protein levels of exosomes and RFVs of PD-1+ or PD-L1+ exosomes in plasma of patients with HNSCC. A. protein concentrations of exosomes (μg/ml plasma) are significantly elevated in patients with active disease (AD) relative to those with no evidence of disease after therapy (NED); B. RFVs for PD-L1+ exosomes is significantly higher in AD patients, patients with positive lymph nodes and patients with the high (III/IV) UICC stage tumors at diagnosis. * p< 0.05; *** p< 0.0008. C. RFVs for PD-1 exosomes in plasma are not statistically different in HNSCC patients stratified by disease activity, lymph node or UICC status. The data presented as box plots are normalized to the frequency of exosomes/1mL of patients’ plasma in this and the subsequent figures.

Article Snippet: Next, anti-PD-1 Ab, #AF1086 lot #ICA02 (purchased from R&D Systems) was used to block effects of exosome-mediated suppression.

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Clinical significance of PD-L1 + exosomes in plasma of Head and Neck Cancer patients

doi: 10.1158/1078-0432.CCR-17-2664

Figure Lengend Snippet: RFVs for PD-L1+ and PD-1+ exosomes/mL plasma and levels of sPD-L1 in plasma of HNSCC patients. A. No correlation between RFVs for PD-L1+ and PD-1+ exosomes in plasma of HNSCC patients was observed: Spearman’s correlation at p=0.7, r=0.05. B. No correlation between RFVs for PD-L1+ exosomes in patients’ plasma and soluble PD-L1; Spearman’s correlation at p=0.85, r=0.03.

Article Snippet: Next, anti-PD-1 Ab, #AF1086 lot #ICA02 (purchased from R&D Systems) was used to block effects of exosome-mediated suppression.

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Clinical significance of PD-L1 + exosomes in plasma of Head and Neck Cancer patients

doi: 10.1158/1078-0432.CCR-17-2664

Figure Lengend Snippet: CD69 and PD-1 expression on activated CD8+ T cells and down-regulation of CD69 expression by PD-L1high but not by PD-L1low exosomes. A. Representative flow cytometry showing expression levels of CD69 or PD-1 on the surface of activated CD8+ T cells. Decreasing levels of CD8+CD69+ on the surface of T cells co-incubated with PD-L1high exosomes. B, Downregulation of CD69 expression (% and MFI) on CD8+ T-cells after co-incubation with PD-L1high exosomes. In C, blocking of exosome-mediated down-regulation of CD69 expression on CD8+ T cells by anti-PD-1 Ab. Note that inhibition mediated by PD-L1high exosomes was almost completely reversed (% and MFI) by adding the PD-1 inhibitor. * p< 0.05; ** p<0.005

Article Snippet: Next, anti-PD-1 Ab, #AF1086 lot #ICA02 (purchased from R&D Systems) was used to block effects of exosome-mediated suppression.

Techniques: Expressing, Flow Cytometry, Incubation, Blocking Assay, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Interleukin-22 is increased in multiple sclerosis patients and targets astrocytes

doi: 10.1186/s12974-015-0335-3

Figure Lengend Snippet: Study subjects for the assessment of IL-22 and IL-22BP in the blood

Article Snippet: “Maxisorp Immunoplates” 96-well plates (Nunc) were coated with coating solution (15 mM Na 2 CO 3 , 34.8 mM NaHCO 3 ) mixed with goat anti-IL-22BP antibody (AF1087, R&D Systems) diluted 1:500 and incubated overnight at 4 °C.

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: Interleukin-22 is increased in multiple sclerosis patients and targets astrocytes

doi: 10.1186/s12974-015-0335-3

Figure Lengend Snippet: IL-22BP and IL-22 are increased in MS patients as compared to healthy controls. The IL-22 and IL-22BP expressions were assessed by ELISA ( a–h ) and qPCR ( i–k ) in the serum ( a , b and f , g ), CSF ( c , h ), isolated monocytes ( i , j ), and moDCs ( k ) and supernatant of SEB-stimulated PBMC for 18 h ( d , e ) isolated from MS patients and healthy controls. Each dot represents a patient. The bars represent the median. Dashed red lines represent the detection limit. Active : clinically active MS patients, inactive : clinically inactive MS patients, progressive : primary and secondary progressive MS patients. Differences among the four groups were significant with Kruskal-Wallis test ( b , e ). Unpaired non-parametric Mann–Whitney test was used to compare groups two-by-two. * P < 0.05, ** P < 0.01

Article Snippet: “Maxisorp Immunoplates” 96-well plates (Nunc) were coated with coating solution (15 mM Na 2 CO 3 , 34.8 mM NaHCO 3 ) mixed with goat anti-IL-22BP antibody (AF1087, R&D Systems) diluted 1:500 and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, MANN-WHITNEY

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Segmental assembly of fibronectin matrix requires rap1b and integrin α5.

doi: 10.1002/dvdy.23909

Figure Lengend Snippet: Fig. 4. Localization of EphrinB2a suggests gradual loss of segment polarity after loss of both rap1b and itga5 function. Dorsal views (A–C:) and higher magnification dorsal views (A’–C’’) of EphrinB2a protein localized to the posterior halves of somites in (A–A’’) wild-type and (B–B’’) rap1b MO1 embryos. Localized EphrinB2a is lost in anterior somites of (C–C’’) itga52/2; rap1b double morphants, but is transiently present in more recently formed posterior somites. Shown are 12–15-somite-stage zebrafish embryos. In A–C, anterior is up, while in A’–C’’ anterior is left.

Article Snippet: Fibronectin and EphrinB2a localization was performed as previously described (J€ulich et al., 2005; Zhang et al., 2008) using rabbit antihuman Fibronectin IgG (Sigma, St. Louis, MO) or goat anti-zebrafish EphrinB2a (R&D Systems, Minneapolis, MN).

Techniques:

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Segmental assembly of fibronectin matrix requires rap1b and integrin α5.

doi: 10.1002/dvdy.23909

Figure Lengend Snippet: Fig. 5. Somite morphogenesis is sensitive to genetic interaction between itga5 and ephrinB2a, but not rap1b and ephrinB2a. A–C: Lateral view DIC images showing complete disruption of somite borders in (A) itga52/2; ephrinB2a MO embryos (7 experiments, 27.9% affected compared to an expected 25% for homozygotes, n ¼ 605) and proper maintenance of somite borders in (B) ephrinB2a MO þ rap1b MO1 (5 experiments, no synergy, n ¼ 455) and (C) ephrinB2a MO þ rap1b DN embryos (3 experiments, no synergy). Dorsal (D–F:) and higher magnification dorsal (D’–F’) views of Fibronectin localization for (D–D’) itga52/2; ephrinB2a MO, (E–E’) ephrinB2a MO þ rap1b MO1, and (F–F’) ephrinB2a MO þ rap1b DN embryos at the 12–15-somite stage. FN matrix formation is lost only in itga52/2; ephrinB2a MO embryos. For all images, anterior is left.

Article Snippet: Fibronectin and EphrinB2a localization was performed as previously described (J€ulich et al., 2005; Zhang et al., 2008) using rabbit antihuman Fibronectin IgG (Sigma, St. Louis, MO) or goat anti-zebrafish EphrinB2a (R&D Systems, Minneapolis, MN).

Techniques: Disruption

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Expression of ARTN in colorectal tissues.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Increased expression of ARTN in CRC is associated with poor prognosis. (A) Immunohistochemical analysis of ARTN protein expression in non-cancerous colorectal tissue (left) and colorectal carcinoma tissue (right). Original images were taken at the magnification of X200 under a microscope. The indicated areas (red rectangular) were enlarged for staining details. The red arrowheads indicate the positive staining for ARTN. (B) Kaplan–Meier analysis of the correlation of the immunohistochemistry-determined ARTN protein expression to relapse-free survival (left) and overall survival (right) of colorectal carcinoma patients. Data were collected from 42 out of 89 colorectal carcinoma cases.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Microscopy, Staining, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Correlation between clinicopathological parameters and ARTN expression in a CRC patient cohort.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Forced expression of ARTN enhances oncogenic behaviour of CRC cells. (A) Total cell number of DLD1-vec and DLD1-ARTN cells under normal culture condition (medium with 10%FBS) or serum deprivation condition (medium with 1% FBS). (B) BrdU incorporation assay determined S-phase entry of DLD1-vec and DLD1-ARTN cells cultured under serum deprivation condition for 24 hours. (C) Hoechst 33258 staining determined apoptotic nuclei percentages of DLD1-vec and DLD1-ARTN cells cultured under serum deprivation condition for 24 hours. (D) Soft agar colony formation of DLD1-Vec and DLD1-ARTN cells. The colony numbers were counted and presented as relative percentage change. (E) Foci formation by DLD1-Vec and DLD1-ARTN cells under serum deprivation conditions for 14 days. At the end of the culture period the colonies were fixed and stained with crystal violet (lower panel), which were then dissolved in 10% SDS and quantified at 570nm absorbance. (F) 3D Matrigel growth of DLD1-Vec and DLD1-ARTN cells. Cell viability was measured by AlamarBlue assay and presented as the relative percentage changes. Red arrowheads indicate the colony cell protrusions into the matrix. (G) Colony scattering assay of DLD1-Vec and DLD1-ARTN cells. The numbers of each type of colonies were calculated under microscope and presented as the percentages of the total counted colony numbers. Representative images of the colony morphology of each colony type of DLD1 cells are presented on the right. Scale bar, 100µm. (H) F-actin stained with Rhodamine-Phalloidin in the DLD1-Vec and DLD1-ARTN cells. The primary images (upper panel) were taken with confocal microscope at 1000X magnification and zoomed in at the area in the white square as shown in the lower panel. Scale bar, 20µm. (I) Wound-healing assay by DLD1-Vec and DLD1-ARTN cells under serum deprivation conditions for 24 hours. The edges of the wound scratches were indicated with dotted guidelines. (J) Transwell migration assay of DLD1-Vec and DLD1-ARTN cells. (K) Transwell invasion assay of DLD1-Vec and DLD1-ARTN cells. * p < 0.05, ** p < 0.01.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Expressing, BrdU Incorporation Assay, Cell Culture, Staining, Alamar Blue Assay, Scattering Assay, Microscopy, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Depletion of ARTN inhibits oncogenic behaviors of CRC cells. (A) Total cell number of DLD1-psilencer and DLD1-siARTN cells under normal culture condition (medium with 10%FBS) or serum deprivation conditions (medium with 1% FBS). (B) BrdU incorporation assay determined S-phase entry of DLD1-psilencer and DLD1-siARTN cells cultured under serum deprivation conditions for 24 hours. (C) Hoechst 33258 staining determined apoptotic nuclei percentages of DLD1-psilencer and DLD1-siARTN cells cultured under serum deprivation condition for 24 hours. (D) Soft agar colony formation of DLD1-psilencer and DLD1-siARTN cells. The colony numbers were counted and presented as relative percentage change. (E) 3D Matrigel growth of DLD1-psilencer and DLD1-siARTN cells. Cell viability was measured by AlamarBlue assay and presented as the relative percentage changes. (F) Transwell migration assay with DLD1-psilencer and DLD1-siARTN cells. (G) Transwell invasion assay with DLD1-psilencer and DLD1-siARTN cells. (H, I) , Western blot analysis for the expression of epithelial and mesenchymal cell markers and EMT regulator SNAIL in the stable cell lines with forced expression (H) or depletion (I) of endogenous ARTN. * p < 0.05, ** p < 0.01.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: BrdU Incorporation Assay, Cell Culture, Staining, Alamar Blue Assay, Transwell Migration Assay, Transwell Invasion Assay, Western Blot, Expressing, Stable Transfection

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Real-time PCR for the mRNA expression of genes in DLD1 cell lines with either forced or depleted expression of ARTN.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: ARTN promotes DLD1 xenograft growth. (A, D) , Subcutaneous tumor growth by DLD1 cells with stable forced expression of ARTN (A) or depletion of endogenous ARTN (D) . Cells were injected subcutaneously to immunocompromised mice. The tumor volume was measured once per week. The significance of differences in the tumor volume was analyzed by ANOVA. The mice were sacrificed when the first tumor volume reached 1500mm 3 and the tumors were harvested. (B, E) , Ki67 staining to determine cell proliferation in the tumors. (C, F) , TUNEL assay to determine apoptosis in the tumors. Results were presented in fold change. ** p < 0.01, *** p < 0.001.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Expressing, Injection, Staining, TUNEL Assay

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: ARTN promotes metastasis of DLD1 cells. (A, D, G, H) , Lung and liver sections from nude mice with tail vein injection of DLD1 cells with stable forced or depleted expression of ARTN (n = 6). The mice were sacrificed 6 weeks after injection and the lung and liver from each mouse were harvested. H&E staining was performed on the paraffin fixed lung (A, D) and liver (G, H) sections. The images were taken under a microscope with magnification of 200X. The arrows indicate established metastatic colonies. (B, E) , Numbers of the metastatic colonies in each lung section. (C, F) , The expression of human HPRT gene in the lung sections was quantified by qPCR with specific primers and normalized against the mouse β-ACTIN . Results were presented in fold change. ** p < 0.01, *** p < 0.001.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Injection, Expressing, Staining, Microscopy

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: ARTN enhanced stem cell-like properties in DLD1 and HCT116 cells. (A) Serial colonosphere formation assay with DLD1 with either stable forced (left) or depleted (right) expression of endogenous ARTN. The colonospheres were passaged successively twice (P1-P3). For each passage of the colonospheres, the initial singular 100 cells were seeded in each well with triplicates and cultured for 7 days before re-seeding. The number of colonospheres (≥100µm in diameter) in each well was counted and presented as the average of the triplicate. The images of the P1 colonospheres were captured under a microscope. Scale bar, 100 µm. (B) Western blotting analysis for the expression of ARTN in the whole cell lysate of monolayer culture or colonospheres of DLD1 cells. (C) , ALDEFLUOR (ALDH1 activity) assay on DLD1 and HCT116 cells with stable forced expression of ARTN (left) or depletion of endogenous ARTN (right). * p < 0.05, ** p < 0.01.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Tube Formation Assay, Expressing, Cell Culture, Microscopy, Western Blot, Activity Assay

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Real time-PCR analysis for mRNA expression of colorectal CSC marker genes and the ARTN gene in DLD1 cells.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Marker

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: Real time-PCR analysis for mRNA expression of CSC markers in DLD1 cells with either forced or depleted expression of ARTN.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: ARTN stimulates its functions through p44/42 MAPK/CDH2 signaling in CRC cells. (A) Western blot analysis for the expression and phosphorylation of p44/42 MAPK in DLD1 cells with stable forced expression or depletion of endogenous ARTN. (B) DLD1 cell pairs were transiently transfected to deplete the endogenous CDH2 (sictrl/siNcad) or to force the expression of CDH2 (ctrl/Ncad), and examined for the expression of CDH2, p44/42 MAPK and ARTN by Western blotting. (C, E) , DLD1 cell pairs were transiently transfected to deplete the endogenous CDH2 (sictrl/siNcad) or to acquire forced expression of CDH2 (ctrl/Ncad), and examined for 3D Matrigel growth (C) , cell invasion (D) , and the population of ALDH1 bright cells (E, F) Western blotting analysis for the expression of CDH2, p44/42 MAPK and ARTN in U0126 treated (5µM for 6 hours) DLD1 cells with stable forced expression of ARTN. (G) Invasion of DLD1 cells with stable forced expression of ARTN in the absence or presence of 5µM U0126. (H) 3D Matrigel growth of DLD1 cells with stable forced expression of ARTN in the absence or presence of 5µM U0126 determined for cell viability by Alamar Blue assay. * p < 0.05, ** p < 0.01.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Phospho-proteomics, Transfection, Alamar Blue Assay

Journal: Frontiers in Oncology

Article Title: ARTEMIN Promotes Oncogenicity and Resistance to 5-Fluorouracil in Colorectal Carcinoma by p44/42 MAPK Dependent Expression of CDH2

doi: 10.3389/fonc.2021.712348

Figure Lengend Snippet: ARTN in CRC cells decreases 5-FU sensitivity and mediates 5-FU resistance. (A) Western blot analysis for the expression of ARTN, CDH2 and p44/42 MAPK in the control (WT) and 5-FU-R cells. (B) Foci formation of 5-FU-R/WT cells in the absence or presence of 3.5µM 5-FU for 7 days. (C) 3D Matrigel growth of 5-FU-R/WT cells in the absence or presence of 3.5µM 5-FU. (D, E) , WT and 5-FU-R cells with transient depletion of ARTN by siRNA were examined for the sensitivity to 5-FU by monolayer proliferation (D) and 3D Matrigel growth (E) . Cell viability was determined by AlamarBlue assay, normalized against that of control WT DLD1 cells (WT siVec) and presented as relative percentages. (F, G) , WT and 5-FU-R DLD1 cells were transiently transfected with specific siRNA against CDH2 and examined for the sensitivity to 5-FU by monolayer proliferation (F) and 3D Matrigel growth (G) . Cell viability was determined by AlamarBlue assay. (H) WT and 5-FU-R DLD1 cells were treated with 5µM U0126 and examined for monolayer proliferation in the absence or presence of 3.5µM 5-FU and determined for cell viability by AlamarBlue assay. * p < 0.05, ** p < 0.01.

Article Snippet: Western blot analysis was performed as described previously ( – ) using the following antibodies: goat ARTN polyclonal antibody (R&D Systems, Minneapolis, MN, USA), mouse β-ACTIN monoclonal antibody (Sigma, St Louis, MO, USA), rabbit phosphorylated ERK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), mouse CDH2 monoclonal antibody (Abcam), RET, phosphorylated RET antibody, Ras antibody and GFRα1 antibody, GFRα3 antibody, mouse anti-β-ACTIN monoclonal antibody (Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Control, Alamar Blue Assay, Transfection