Journal: PLoS ONE

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent

doi: 10.1371/journal.pone.0128926

Figure Lengend Snippet: (A) RAW264.7 cells were stimulated with recombinant F-spondin proteins (rF-spondin) for 24 h and IL-1Ra production was measured by ELISA; **, P < 0.01. (B) RAW264.7 cells were transfected with either Myc-tagged F-spondin plasmid (pF-spondin) or a control vector (VC), and incubated for 24h. The success of transfection was verified by western blotting analysis of F-spondin and Myc expression. The IL-1Ra production was measured by ELISA 48h after transfection; **, P < 0.01. (C) Effect of F-spondin knockdown on ODN1668-induced IL-1Ra expression. Knockdown of F-spondin was verified by RT-PCR analysis. Wild-type RAW264.7 cells (RAW WT ) and RAW264.7 cells stably transfected with F-spondin shRNA (RAW spondin-shRNA ) or scrambled shRNA (RAW Mock-shRNA ) were incubated with or without 1 μM ODN1668 for the indicated times. RT-PCR was then performed to analyze IL-1Ra expression. β-actin was used as a loading control. (D) Bone marrow derived macrophages were transfected with scrambled siRNA or siRNA for F-spondin gene. After cells were attached to the plate, media were removed and fresh media containing 6 μM ODN1668 were supplied. The expressions of F-spondin and IL-1Ra were examined after 1d treatment. Similar results were obtained in two independent experiments. (E) The IL-1Ra production of cells in (D) was measured by ELISA; **, P < 0.01. Data shown are average of three independent experiments.

Article Snippet: Anti-CDK2 and anti-actin antibodies as well as siRNAs for F-spondin and IL-1Ra genes were obtained from Santa Cruz Biotechnology (St. Louis, MO).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Expressing, Knockdown, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, shRNA, Derivative Assay

Journal: PLoS ONE

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent

doi: 10.1371/journal.pone.0128926

Figure Lengend Snippet: (A) RAW264.7 cells treated with or without 5 μg/ml recombinant F-spondin protein (rF-spondin) were cultured in complete media for 48 h and assayed for viability; ***, P < 0.001. (B) RAW264.7 cells treated with or without 1 μM ODN1668 and IL-1Ra overexpressing stable cell line (RAW IL-1Ra ) were cultured in complete media for 48 h and assayed for viability; ***, P < 0.001. Overexpression of IL-1Ra in RAW IL-1Ra was confirmed by RT-PCR. (C) Wild-type RAW264.7 cells (WT), RAW264.7 cells stably transfected with empty vector (VC), and RAW IL-1Ra cells (RA) were plated at the same density and cultured in complete media for 48 h. Cell cycle analysis was performed by flow cytometry and the data were analyzed using ModFit software. Results shown are representative of three independent experiments; **, P < 0.01. (D) The expression of cell cycle-related protein in RAW VC and RAW IL-1Ra cells were examined by western blotting. (E) Bone marrow derived macrophages (BMMs) were transiently transfected with a control vector (VC) or IL-1Ra plasmid (RA). The expressions of IL-1Ra and p27 were examined 24h after transfection. (F) Western blotting analysis of IL-1Ra and p27 expressions in BMMs transfected with scrambled siRNA or siRNA for IL-1Ra gene. Cells were incubated with 6 μM ODN1668 for 24h before harvest.

Article Snippet: Anti-CDK2 and anti-actin antibodies as well as siRNAs for F-spondin and IL-1Ra genes were obtained from Santa Cruz Biotechnology (St. Louis, MO).

Techniques: Recombinant, Cell Culture, Stable Transfection, Over Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Cell Cycle Assay, Flow Cytometry, Software, Expressing, Western Blot, Derivative Assay, Control, Incubation

Journal: PLoS ONE

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent

doi: 10.1371/journal.pone.0128926

Figure Lengend Snippet: Trajectories of cells in response to different treatments were measured by time-lapse microscopy and displayed in diagrams drawn with the initial point of each trajectory placed at the origin of the plot. Serial phase-contrast images were obtained every 15 min for 22 h by time-lapse microscopy. Trajectories (upper panel) and migration velocity (lower panel) of cells were displayed after 22 h of time-lapse. Results of migration velocities represent data of 20 cells (means ± SEM). (A) RAW264.7 and J774A.1 cells were treated with or without 6 μM ODN1668 or 6 μM GpC ODN1668 (ODN1668GC). (B) RAW264.7 and J774A.1 cells were treated with or without 2 μg/ml recombinant F-spondin protein (rF-spondin). (C) RAW Mock-shRNA and RAW Spondin-shRNA stable cell lines were treated with or without 6 μM ODN1668. (D) The trajectory and migration velocity of untreated RAW VC and RAW IL-1Ra stable cell lines were compared. (E) RAW264.7 cells were transfected with scrambled siRNA or siRNA for IL-1Ra gene. After cells were attached to the plate, media were removed and fresh media containing 6 μM ODN1668 were supplied. The transfection efficiency was verified by western blotting. *, P < 0.05, ****, P < 0.0001, N.S., not significant.

Article Snippet: Anti-CDK2 and anti-actin antibodies as well as siRNAs for F-spondin and IL-1Ra genes were obtained from Santa Cruz Biotechnology (St. Louis, MO).

Techniques: Time-lapse Microscopy, Migration, Recombinant, shRNA, Stable Transfection, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent

doi: 10.1371/journal.pone.0128926

Figure Lengend Snippet: BMMs were transfected with scrambled siRNA or siRNA for IL-1Ra and/or F-spondin gene. After cells were attached to the plate, media were removed and fresh media containing 6 μM ODN1668 were supplied. Trajectories (upper panel) and migration velocity (lower panel) of cells were displayed after 22 h of time-lapse as described in the Materials & Methods section. **, P < 0.01, ****, P < 0.0001.

Article Snippet: Anti-CDK2 and anti-actin antibodies as well as siRNAs for F-spondin and IL-1Ra genes were obtained from Santa Cruz Biotechnology (St. Louis, MO).

Techniques: Transfection, Migration

Journal: JCI insight

Article Title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Figure 1. Identifying Spon1 as an important mediator of LUSC disease progression. (A) Model showing the development of the KAL-LN2E1 metastatic LUSC cell line by in vivo passaging. (B) RNA-Seq data of genes that are differentially expressed in TIMs from both the LN2E1 and LN4K1 tumor models (across the 3 experimental conditions) when compared with their respective host strain BM-derived IMs (used as baselines). (C) Relative expression of top 10 genes with highest hazard ratios found to be overexpressed in TIMs by qPCR. (D) Spon1 expression in healthy versus tumor-bearing IMs from tumors and blood. (E) Plasma SPON1 concentrations (ng/mL) in healthy versus tumor bearing mice. (F) measure of SPON1 released from WT versus Spon1–/– IMs. (G) SPON1 levels as seen in plasma taken from WT versus Spon1–/– mice with orthotopic LLC tumors at day 19. (H) Total SPON1 expression for each cell type from human NSCLC single cell samples. ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Biomarker Discovery, In Vivo, Passaging, RNA Sequencing, Derivative Assay, Expressing, Clinical Proteomics, Two Tailed Test

Journal: JCI insight

Article Title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Figure 3. Spon1 increases fibrillar collagen and collagen remodeling gene expression in cancer cells. (A) Schematic and representative LN2E1 spheroid formation assay in Matrigel with recombinant SPON1 treatment. (B and C) Spheroid area and numbers for LLC and spheroid area and numbers for LN2E1 with and without recombinant murine SPON1 treatment (5 μg/mL). (D and E) Collagen gene expression with SPON1 treatment in LLC spheroids and in LN2E1 spheroids. (F) Schematic of WT or Spon1–/– IM coculture with LN2E1 or LLC spheroids. (G) Spheroid area and collagen gene expression with WT or Spon1–/– IM coculture with LN2E1 spheroids. (H) Collagen gene expression with WT or Spon1–/– IM coculture with LLC spheroids. (I) Collagen gene expression of LN2E1 and LLC spheroids with recombinant SPON1, WT IMs, Spon1–/– IMs, or recombinant SPON1 plus Spon1–/– IMs. ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Gene Expression, Tube Formation Assay, Recombinant, Two Tailed Test

Journal: JCI insight

Article Title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Figure 4. Reduced tumor formation and collagen production in the absence of Spon1. (A) IVIS imaging at 15 days from injection of LLC tumors into either WT or Spon1–/– mice. (B) Three-dimensional optical plus CT imaging at day 15 of LLC tumors in WT and Spon1–/– mice with higher resolution quantification. (C) Weight and counts of tumor burden from LLC tumors in WT and Spon1–/– mice. (D) Picrosirius red staining of FFPE tumors from WT and Spon1–/– mice. (E) Quantification of collagen read outs for high-density matrix (HDM). (F) Schematic of CD8 depletion experiment. (G) IVIS results at day 13 of LLC tumors in WT or Spon1–/– mice with Isotype or anti-CD8 antibody treatment. ***P < 0.001, **P < 0.01, *P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons. Total original magnification, ×20.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Imaging, Injection, Staining, Two Tailed Test

Journal: JCI insight

Article Title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Figure 5. Phenotypic rescue of disease burden with restoration of Spon1+ TIMs. (A) Schematic of adoptive transfer experiment with either WT or Spon1–/– IMs infused into Spon1–/– mice orthotopically injected with LLC cells. Infusions took place on days 6, 8, 11, and 12 after injections. (B) IVIS results on day 14 after injection. n = 3. (C) Gross histology visualization of disease burden. (D) Lymph node tumor weight and counts on day 14 after injection. n = 3. (E) Representative IVIS images of WT and Spon1–/– mice with or without adoptive transfer of WT or KO IMs. (F) IVIS results on day 5 after injection. n = 7 each group. (G) IVIS results on day 13 after injection. n = 7 each group. **P < 0.01, *P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Adoptive Transfer Assay, Injection, Two Tailed Test

Journal: JCI insight

Article Title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Figure 6. Spon1 mediates its effects on disease progression and collagen content via LRP8. (A and B) Collagen gene expression in both LN2E1 (A) and LLC (B) spheroids for negative control and LRP8 KO. (C and D) In vivo disease burden by tumor weight and counts in LN2E1 and LLC. (E) Collagen content in LLC shown by Sirius red high-density matrix levels. (F) In vivo IVIS results of LLC WT or LRP8-KO tumors in either WT mice or Spon1–/– mice. n = 9 each group. (G) TGF-β1 as a top upstream regulator of the genes composing our Collagen Gene Signature. (H and I) Phospho-SMAD2 scoring of WT and Spon1–/– LLC tumors and of negative control and LRP8-KO LLC tumors. Scale bars: 125 μM. (J) qPCR of Collagen and EMT genes of LN2E1 WT or LRP8-KO spheroids under treatment conditions of untreated, recombinant SPON1, TGF-βi (SB431542), recombinant SPON11+TGF-βi, and KRFK peptide. ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Biomarker Discovery, Gene Expression, Negative Control, In Vivo, Recombinant, Two Tailed Test

Journal: JCI insight

Article Title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Figure 7. Patient NSCLC tumors show Spon1+ TIMs, and LRP8 expression on cancer cells and TGF-β1 signaling positively correlate with collagen expression, which leads to poor survival. (A) CCR2+SPON1+ staining identifies SPON1+ TIMs in tumor cell islets (PanCK+) in an LUAD tumor. PanCK, white; CCR2, red; SPON1, green; Hoechst (nuclear stain), blue. Scale bar: 25 μM. Arrowheads are pointing to a CCR2+SPON1+ TIM. (B) Immunofluorescence staining of LUSC tumors showing cancer cells (aqua) expressing LRP8 (green) with TGF-β1 activation (+pSMAD2/red). Scale bars: 300 μM (full core), 50 μM (insets). (C) Sirius red staining of the same core used in B and an overlay with positive sirius red staining shown in white. (D and E) Two-sided Pearson correlations of percentage of pSMAD2+L- RP8+panCK+ cells with CCR2+SPON1+ cell density (P < 0.0001, r = 0.272) (D) and between pSMAD2+LRP8+panCK+ cells and high-density matrix indices based on Sirius red staining (E) (P = 0.008, r = 0.139). (F) Survival differences between patients expressing high and low HDM (n = 164 patients, P = 0.0057).

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Expressing, Staining, Immunofluorescence, Activation Assay

Journal: Biochemical and biophysical research communications

Article Title: Effect of F-spondin on cementoblastic differentiation of human periodontal ligament cells.

doi: 10.1016/j.bbrc.2006.08.142

Figure Lengend Snippet: Fig. 2. F-spondin expression in human periodontal tissues. (A) Checking the accuracy of the anti-SPON1 antibody by Western blot. F-spondin antibody specifically recognizes F-spondin in FLAG-F-spondin transfected 293T cells. (B) Immunohistochemical staining with the anti-SPON1 antibody in human periodontal tissue. High expression of F-spondin was observed in cell located along the root surface (triangles). C, cementum; D, dentin; PL, periodontal ligament; AB, alveolar bone. Original magnification ·40 (a), ·200 (b).

Article Snippet: To examine expression of F-spondin in seven human periodontal tissues cases, the 4.5 lm sections were stained immunohistochemically with an anti-SPON1 antibody (Prosci).

Techniques: Expressing, Western Blot, Transfection, Immunohistochemical staining, Staining

Journal: Alzheimer's & Dementia

Article Title: Assessing Aβ‐independent effects of Module 42 on immune function in vitro

doi: 10.1002/alz.71215

Figure Lengend Snippet: Assessing the effect of M42 recombinant proteins on intracellular Ca 2+ signaling in human induced pluripotent stem cell‐derived macrophages. (A) Summary graph showing the fold change in total Ca 2+ levels following the addition of recombinant M42 proteins. (B–G) Intracellular Ca 2+ levels following treatment with TMEFF2 ectodomain (B), MDK (C), SDC4 ectodomain (D), SFRP1 (E), SPON1 (F), and PTN (G). Left panels represent intracellular Ca 2+ levels defined as area under the curve (AUC). Right panels show representative traces from one biological repeat (n = 3 technical replicates). Intracellular Ca 2+ levels are measured as relative fluorescent units. Histamine (6 µM) was used as a positive control. N = 3 to 6 independent biological experiments (two independent macrophage factory set‐ups; four technical replicates/biological repeat). Error bars correspond to mean ± SD. Repeated measures one‐way ANOVA followed by Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.01.

Article Snippet: These include HTRA1 (2916‐SE‐020, Novus Biologicals; E. coli expression system), Fms‐related tyrosine kinase 1 (FLT1; FCL1058B, G&P Biosciences; mammalian expression system), SPON1 (3135‐SP‐025/CF, R&D Systems; mammalian expression system), GPC5 (2607‐G5‐050/CF, R&D Systems; mammalian expression system), and Olfactomedin‐like 3 (OLFML3; Acro Biosystems, OL3‐H52H4‐100ug; mammalian expression system).

Techniques: Recombinant, Derivative Assay, Positive Control

Journal: Alzheimer's & Dementia

Article Title: Assessing Aβ‐independent effects of Module 42 on immune function in vitro

doi: 10.1002/alz.71215

Figure Lengend Snippet: Assessing the effect of M42 protein treatment on viability of hiPSC derived macrophages. Human iPSC macrophages were treated with HTRA1 (A), DAG1 ectodomain (B), TMEFF2 ectodomain (C), MDK (D), GPC5 (E), SDC4 ectodomain (F), SFRP1 (G), SPON1 (H), and PTN (I) at 3 µg/mL, 1 µg/mL, and 100 ng/mL for 24 h. Viability was assessed and expressed as relative luminescence units (RLU). N = 2 or 3 independent biological experiments (one independent macrophage factory set‐up; three technical replicates/biological repeat). Error bars correspond to mean ± standard deviation (SD). Repeated measures one‐way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: These include HTRA1 (2916‐SE‐020, Novus Biologicals; E. coli expression system), Fms‐related tyrosine kinase 1 (FLT1; FCL1058B, G&P Biosciences; mammalian expression system), SPON1 (3135‐SP‐025/CF, R&D Systems; mammalian expression system), GPC5 (2607‐G5‐050/CF, R&D Systems; mammalian expression system), and Olfactomedin‐like 3 (OLFML3; Acro Biosystems, OL3‐H52H4‐100ug; mammalian expression system).

Techniques: Derivative Assay, Standard Deviation