Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 1. Lack of expression of OPN suppresses tumor growth and reduces microvessel density by attenuating the infiltration of COX-2- positive macrophages. (A) Tumor growth kinetics was plotted as tumor volume vs time in weeks (left panel), tumor weights were measured and represented as bar graph (right panel). Error bars represent s.e.m. for tumor volume and s.d. for tumor weight, *Po0.001 versus Opn þ / þ. Six mice were used in each set of experiment. (B) Immunohistochemical staining of tumor sections with anti-CD31 (red; a, b), anti-F4/80 (green; c, d), anti-CD11b (green; e, f), and in combination of anti-F4/80 (green) with anti-COX-2 (red) (double positive, yellow, g, h) antibodies. (C) Tumor growth kinetics of control and Etoricoxib-treated mice: tumor volume vs time in weeks was plotted (left panel). Tumor weights were measured and represented as bar graph (right panel). Error bar represents s.e.m. for tumor volume and s.d. for tumor weight, *Po0.001 versus control. (D) Immunohistochemical staining of tumor sections with anti-CD31 (red; a, b), anti-F4/80 (green; c, d) and anti-CD11b (green; e, f) antibodies. (E) Level of serum PGE2 in Opn þ / þ and Opn / mice (left panel) and control and Etoricoxib-treated mice (right panel) was determined by PGE2 EIA and represented in the form of bar graph. Error bar represents s.e.m., *Po0.001 versus Opn þ / þ and *P ¼ 0.002 versus control.

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Control

Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 2. Co-culture and co-migration of macrophages (RAW264.7) with melanoma (B16F10 and A375) cells upregulate OPN and COX-2 expression. (a) RAW264.7 cells were supplemented with CM of melanoma. Expressions of OPN and COX-2 were analyzed by western blot. Actin was used as loading control (upper and lower panels). PGE2 was estimated by EIA and represented in the form of bar graph, *P ¼ 0.008 (right panel). (b) B16F10 cells were supplemented with CM of RAW264.7 and expressions of OPN and COX-2 were analyzed by western blot. (c) RAW264.7 (1 106) cells either alone or transfected with OPNi, COX-2i, OPN cDNA or COX-2 cDNA were used in the upper chamber, whereas CM of melanoma was added in the lower chamber. Migrated cells were stained, photographed (left panel) and migration of RAW264.7 cells in response to CM of B16F10 were quantified and represented in form of bar graph. The error bar represents s.e.m., *Po0.001 versus Med; ##Po0.001 versus CM of B16F10. **Po0.005 versus CM of B16F10 (right panel). All data are representation of three experiments. (d) Expressions of OPN and COX-2 in cells transfected with their specific siRNA were analyzed by western blot. Actin was used as control (left and right panels).

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Co-Culture Assay, Migration, Expressing, Western Blot, Control, Transfection, Staining

Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 3. OPN upregulates COX-2 expression and COX-2-dependent PGE2 production in an autocrine manner in macrophages via a9 integrin. (a) RAW264.7 cells were supplemented with CM of melanoma (B16F10 or A375), pre-incubated with OPN neutralizing antibody (5 mg/ml) and expression of COX-2 was analyzed by western blot (upper panel). RAW264.7 cells were supplemented with CM of melanoma (B16F10 or A375) cells transfected with OPN siRNA (60 pmol/ml). COX-2 expression was analyzed by western blot (lower panel). Actin was used as loading control. (b) RAW264.7 cells were transfected with OPN siRNA (60 pmol/ml) and then treated with CM of B16F10 and expressions of COX-2 and OPN were analyzed by western blot (upper panel). Peritoneal macrophages (pMac) isolated from Opn þ / þ (^ þ / þ) and Opn / (^ / ) mice were treated with CM of B16F10 and COX-2 expression was analyzed by western blot (lower panel). (c) RAW264.7 cells or pMac were treated with various doses of OPN and COX-2 expression was analyzed by western blot (upper and lower panels). (d) RAW264.7 cells were transfected with OPN cDNA, OPN siRNA (60 pmol/ml) or OPN siRNA-transfected cells were cotransfected with OPN cDNA or treated with human or mouse OPN. The expressions of COX-2 and OPN were analyzed by western blot (left panel). RAW264.7 cells were treated with OPN (0-1 mM). The level of PGE2 was estimated by EIA and represented in the form of bar graph. The error bar represents s.e.m., *Po0.05 versus control (right panel). The data represent three experiments exhibiting similar results. (e) RAW264.7 cells were transfected with a9 integrin siRNA (40 pmol/ml) or pretreated with RGD (10 mM) or control peptide (10 mM) and then treated with OPN (0.5 mM) and COX-2 expression was analyzed by western blot (upper panel). Expression of a9 integrin in cells transfected with its specific siRNA was analyzed (lower panel). Actin was used as loading control.

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Incubation, Western Blot, Transfection, Control, Isolation

Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 4. OPN-activated macrophages trigger ICAM-dependent HUVEC migration and promote tube formation via PGE2. (a) RAW264.7 cells, either treated with OPN alone or pretreated with NS-398 (10 mM), Etoricoxib (10 mM) and then treated with OPN (0.5 mM), were used in the lower chamber, whereas HUVEC were used in the upper chamber. HUVEC migrated toward reverse side of the upper chamber were stained and photographed (left panel). Migration of HUVEC in response to CM of OPN-activated macrophages were quantified and represented in form of bar graph; Error bars, ±s.e.m., *P ¼ 0.025 versus Med; **Po0.001 versus untreated macrophages; #Po0.001 versus CM of OPN-activated macrophages (right panel). (b) HUVEC were supplemented with CM of RAW264.7 cells treated with OPN either alone or along with NS-398, Etoricoxib or transfected with COX-2 siRNA or OPN cDNA. ICAM expression was analyzed by western blot (upper panel). HUVEC were treated with PGE2 and expression of ICAM was analyzed by western blot (lower panel). (c) Wounds were made and HUVEC were pretreated with two doses of ICAM blocking antibody and then treated with PGE2. Wound photographs were taken after 12 h. (d) HUVEC (1 104) were seeded on matrigel-coated plate and then supplemented with CM of RAW264.7 cells either treated with OPN alone or pretreated with NS-398, Etoricoxib and then treated with OPN (0.5 mM). PGE2 was used as positive control. After 4 h of incubation, endothelial cell tubular structure formation was photographed and analyzed.

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Staining, Transfection, Expressing, Western Blot, Blocking Assay, Positive Control, Incubation

Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 5. OPN-activated macrophages augment pro-angiogenic activity via COX-2 in CAM assay. (a) The CAM of fertilized white leghorn chicken eggs was added with uniform-sized sterilized gelatin sponges. The sponges were loaded with 20 ml of CM from RAW264.7 cells treated with either OPN alone (0.5 mM) or pretreated with NS-398, Etoricoxib or vehicle control (DMSO) and then treated with OPN (0.5 mM). CAM was photographed in ovo with a stereomicroscope equipped with a camera. PGE2 was used as a positive control. (b, c) At day 6, the angiogenic response was evaluated as the length or size of vessels converging toward the sponge and angiogenic activity was quantified using Angioquant Software. The error bar represents s.e.m., *P ¼ 0.008 versus Med; ##P ¼ 0.007 versus CM of OPN-activated macrophage.

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Chick Chorioallantoic Membrane Assay, Control, In Ovo, Positive Control, Software

Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 6. OPN induces ERK1/2- and p38-dependent AP-1-mediated COX-2 expression and COX-2-dependent MMP-9 expression in macrophages. (a) RAW264.7 cells were treated with OPN in a dose (0–1 mM) -dependent manner. Expression of CD206 (mannose receptor) was analyzed by western blot. Actin was used as loading control. (b) RAW264.7 cells were treated with OPN (0.5 mM) in time (0–60 min) -dependent manner and the level of phospho-ERK1/2 and phospho-p38 were analyzed by western blot. The blots were reprobed with anti-ERK and anti- p38 antibodies. (c) RAW264.7 cells were pretreated with SB203580 (10 mM) or PD98059 (25 mM) and then treated with OPN (0.5 mM) and COX-2 expression was analyzed by western blot (upper panel). In separate experiments, cells were pretreated with inhibitor of p38 (SB203580, 10 mM), ERK (PD98059, 25 mM) or COX-2 (NS-398, 10 mM; Etoricoxib, 10 mM) and then treated with OPN (0.5 mM). The level of PGE2 was estimated by EIA and represented in the form of bar graph. The error bar represents s.e.m., *Po0.001 versus Med; ##Po0.001 versus OPN (lower panel). (d) RAW264.7 cells were treated with OPN (0.5 mM) in a time (0–240 min) -dependent manner. The expression of c-Fos and c-Jun were analyzed by western blot. Actin was used as loading control. (e) Cells were treated with OPN (0.5 mM) for 0–240 min and nuclear localization of c-Fos was detected by immunofluorescence using anti-c-Fos antibody followed by stained with Cy3 (red). Nuclei were stained with DAPI (blue). (f) RAW264.7 cells were pretreated with SB203580 or PD98059 and then treated with OPN (0.5 mM). The expression and nuclear localization of c-Fos was detected by immunofluorescence using anti-c-Fos antibody, stained with Cy3 (red) and nuclei were stained with DAPI (blue) (left panel). RAW264.7 cells were transfected with Wt c-Jun, DN c-Jun or A-Fos cDNA and then treated with OPN (0.5 mM) and COX-2 expression was analyzed by western blot. Actin was used as loading control (right panel). (g) RAW264.7 cells were treated with OPN in a dose (0-1 mM) -dependent manner and MMP-9 expression in CM was detected by zymography (upper panel) and western blot (lower panel). RAW264.7 cells were pretreated with inhibitors SB203580, PD98059 or NS-398 and then treated with OPN and MMP-9 expression in CM was detected by zymography (right panel). All data are representation of three experiments exhibiting similar results.

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control, Staining, Transfection, Zymography

Journal: Oncogene

Article Title: Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin.

doi: 10.1038/onc.2013.184

Figure Lengend Snippet: Figure 7. OPN-activated macrophages induce melanoma cell migration. (A) RAW264.7 cells treated with either OPN alone or pretreated with NS-398 or Etoricoxib and then treated with OPN and used in lower chamber, whereas melanoma cells were used in the upper chamber. After 12 h, migrated cells were stained and photographed (left panel). Bar graph represents the migration of melanoma cells toward OPN-activated macrophage. The error bar represents s.e.m., *Po0.001 versus untreated (Un) macrophages, ##Po0.001 versus OPN-activated macrophages (right panel). (B, C) Infiltrating macrophages (CD68 positive) in malignant melanoma are OPN and COX-2 positive and correlates with tumor angiogenesis. Histopathological (H&E; a, b) and immunohistochemical staining of peripheral normal and malignant melanoma specimens with anti-CD68 (red; c, d), co-staining with anti-CD68, anti-OPN and DAPI (e, f; white arrow indicates OPN-positive macrophages) and anti- CD31 (red; g, h). (C) Colocalization of COX-2 (green) and macrophages (CD68, red) was observed in normal peripheral and malignant melanoma specimens (a–h). (D) Schematic representation of tumor-educated OPN-activated macrophages via a9 integrin regulates ERK- and p38-dependent AP-1 activation leading to enhanced COX-2 expression and PGE2 production, which in turn upregulates melanoma growth and angiogenesis.

Article Snippet: Cell lines and transfections The mouse macrophage (RAW264.7, IC21) and melanoma (B16F10), human melanoma (A375) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Staining, Immunohistochemical staining, Activation Assay, Expressing

Journal: Aging (Albany NY)

Article Title: Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration

doi: 10.18632/aging.102909

Figure Lengend Snippet: Preparation of AAVs that deplete DNMT1 and TGFβ1. ( A ) Schematic to show the structure of AAVs carrying shRNA for DNMT1 (shDNMT1) or shRNA for TGFβ1 (shTGFβ1) under a macrophage-specific CD68 promoter. The control AAV carried a scramble sequence under the CD68 promoter. The constructs were connected to a GFP reporter by p2A to allow co-expression by one promoter. ( B , C ) DNMT1 ( B ) and TGFβ1 ( C ) levels were examined in 3 mouse macrophage lines, Raw264.7, J774A.1 and IC-21, by RT-qPCR. ( D – G ) IC-21 was transduced with different AAVs. DNMT1 levels were determined by RT-qPCR ( D ) and by Western blot ( E ). TGFβ1 levels were determined by RT-qPCR ( F ) and by ELISA ( G ). *p<0.05. NS: non-significant. N=5.

Article Snippet: Several mouse macrophage lines (RAW264.7, J774A.1 and IC-21) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco; Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA), penicillin (100μg/ml) and streptomycin (250ng/ml) at 37°C, in a 5% CO 2 atmosphere.

Techniques: shRNA, Control, Sequencing, Construct, Expressing, Quantitative RT-PCR, Transduction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration

doi: 10.18632/aging.102909

Figure Lengend Snippet: CD68 promoter in AAVs allows specific targeting of macrophages. After LDD was induced in mice, orthotopic injection of these AAVs into LDD-mice was performed. Mice in the scramble group received an orthotopic injection of 100 μl 10 11 AAV-pCD68-scramble; mice in the shDNMT1 group received an orthotopic injection of 100 μl 10 11 AAV-pCD68-shDNMT1; mice in the shTGFβ1 group received an orthotopic injection of 100 μl 10 11 AAV-pCD68-shTGFβ1; mice in the shDNMT1+shTGFβ1 group received an orthotopic injection of 50 μl 0.5X10 11 AAV-pCD68-shDNMT1 and 50 μl 0.5X10 11 AAV-pCD68-shTGFβ1. Mice were kept for 4 weeks before analysis. ( A , B ) F4/80 staining and GFP signals are shown by representative images ( A ) and by quantification of the percentage of GFP+ cells in all F4/80+ cells ( B ). NS: non-significant. N=7. Scale bars are 20μm.

Article Snippet: Several mouse macrophage lines (RAW264.7, J774A.1 and IC-21) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco; Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA), penicillin (100μg/ml) and streptomycin (250ng/ml) at 37°C, in a 5% CO 2 atmosphere.

Techniques: Injection, Staining

Journal: Aging (Albany NY)

Article Title: Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration

doi: 10.18632/aging.102909

Figure Lengend Snippet: Effects of AAVs on cytokine production by macrophages. ( A ) F4/80+ macrophages in the degeneration zone were isolated by flow cytometry, as shown by representative flow charts. ( B – G ) ELISA for TNFα ( B ) IL-1β ( C ) IL-6 ( D ) IL-4 ( E ) IL-10 ( F ) and TGFβ1 ( G ). *p<0.05. NS: non-significant. N=7.

Article Snippet: Several mouse macrophage lines (RAW264.7, J774A.1 and IC-21) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco; Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA), penicillin (100μg/ml) and streptomycin (250ng/ml) at 37°C, in a 5% CO 2 atmosphere.

Techniques: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration

doi: 10.18632/aging.102909

Figure Lengend Snippet: Effects of AAVs on macrophage polarization. ( A , B ) Flow cytometry for CD86 and CD206 on F4/80+ cells from the LDD zone, as shown by representative flow charts ( A ) and by quantification of the ratio of CD206+ M2 macrophages to CD86+ M1 macrophages ( B ). *p<0.05. NS: non-significant. N=7.

Article Snippet: Several mouse macrophage lines (RAW264.7, J774A.1 and IC-21) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco; Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA), penicillin (100μg/ml) and streptomycin (250ng/ml) at 37°C, in a 5% CO 2 atmosphere.

Techniques: Flow Cytometry