Journal: Oncology Reports

Article Title: Effects of CCL5 on the biological behavior of breast cancer and the mechanisms of its interaction with tumor-associated macrophages

doi: 10.3892/or.2019.7344

Figure Lengend Snippet: Morphological analysis and surface marker determination of TAMs induced from THP-1 cells in vitro . (A) THP-1 cell morphology (magnification ×200) and cell morphology following treatment with PMA for 24 h (magnification ×200). Cell morphology was analyzed following treatment with PMA for 24 h, and IL-4 and IL-13 for 18 h (magnification ×200). TAM cell morphology cultured after 24 h (×200) was also analyzed. (B) The expression of CD204 and CD206 on the surface of THP-1 cells before and after induction was investigated. **P<0.01, ***P<0.0001 vs. THP-1. (C) Transwell assay of chemotaxis ability of CCL5 to TAMs (magnification ×200), **P<0.01, ***P<0.001. APC, allophycocyanin; CCL5, C-C motif chemokine ligand 5; IL, interleukin; PMA, phorbol-12-myristate-13-acetate; TAMs, tumor-associated macrophages.

Article Snippet: After protein blocking with 10% goat serum (Abcam) at room temperature for 30 min, sections were incubated with primary antibodies (all 1:100) against human CCL5 (cat. no. P230E; Invitrogen; Thermo Fisher Scientific, Inc.), F4/80, CD16, and CD206 (cat. nos. bs-11182R, bs-6028R and bs-21473R, respectively; Beijing Bioss Biotechnology, Ltd.) overnight at 4°C and rinsed with TBS.

Techniques: Marker, In Vitro, Cell Culture, Expressing, Transwell Assay, Chemotaxis Assay

Journal: Oncology Reports

Article Title: Effects of CCL5 on the biological behavior of breast cancer and the mechanisms of its interaction with tumor-associated macrophages

doi: 10.3892/or.2019.7344

Figure Lengend Snippet: Effects of CCL5 on the growth of human breast cancer xenograft tumor. (A) The xenograft tumor size in nude mice when treated for 30 days. (B) Growth curve of xenograft tumor in nude mice. (C) Nude mice were sacrificed and tumor tissue was obtained on day 30 after administration; the tumor weight was statistically analyzed. *P<0.05, **P<0.01 vs. Control group. (D) The infiltration number of different types of macrophages in human breast cancer xenograft tumor in nude mice (magnification, ×400). F4/80 represents macrophages; CD16 represents M1-type macrophages; and CD206 represents M2-type macrophages. **P<0.01 vs. control group. CCL5, C-C motif chemokine ligand 5.

Article Snippet: After protein blocking with 10% goat serum (Abcam) at room temperature for 30 min, sections were incubated with primary antibodies (all 1:100) against human CCL5 (cat. no. P230E; Invitrogen; Thermo Fisher Scientific, Inc.), F4/80, CD16, and CD206 (cat. nos. bs-11182R, bs-6028R and bs-21473R, respectively; Beijing Bioss Biotechnology, Ltd.) overnight at 4°C and rinsed with TBS.

Techniques:

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Primers for Each Gene for PCR Analysis

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques:

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Exogenous BMP4-induced allodynia, microglial activation and polarization. ( A ) PWT in rats after intrathecal BMP4 application showed a significant effect on time: F (4.143, 66.29) = 16.09, P<0.01, left; F (3.960, 63.35) = 12.67, P<0.01, right, intrathecal application: F (1, 16) = 161.3, P<0.01, left; F (1, 16) = 160.5, P<0.01, right and interaction: F (7, 112) = 11.76, P<0.01, left; F (7, 112) = 5.653, P<0.01, right. Compared with the Sham group, rats in the BMP4 group developed a significant decrease in bilateral PWT for the whole first week, P<0.01, n=9 at each time point for both groups. ( B ) Representative Western blotting showed a sustained increase of CD11b (P<0.01) and CD16 (P<0.01) expressions for the whole 1st week in the BMP4 group compared with the Sham group. Meanwhile, ARG-1 levels increased at day 1 (P<0.01) and day 4 (P<0.01), then fell below the levels of the Sham group (P<0.01). n=3 for each column. ( C and D ) Double-immunofluorescence further detected that compared with the Sham group (P7), CD11b expression was elevated in the dorsal horn of spinal cord after BMP4 treatment. Moreover, expressions of CD16 and ARG-1 showed similar pattern with Western blotting results, which were both mainly accumulated with CD11b + cells. n=3 for each column. Two-way ANOVA, followed by a Bonferroni test ( A ) and one-way ANOVA, followed by Sidak’s multiple comparisons test ( B – D ) were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Exogenous BMP4 induced a distinct pattern of M1/M2 gene expressions. RT-PCR analysis showed that M1-gene markers, including CD16, TNF-α and MHC-II, significantly increased throughout the whole 1st week, while M2-gene markers including ARG-1, CD-204 and IL-4 increased at an early stage (P1 or P4), then all fell to the Sham level at day 7. n=3 for each time point for both groups. One-way ANOVA, followed by Sidak’s multiple comparisons test were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Noggin relieved allodynia, attenuated microglial activation, and changed the polarized pattern after SNL. ( A ) PWT in rats receiving intrathecal Noggin application after SNL showed a significant effect on time: F (3.463, 55.41) = 162.3, P<0.01, intrathecal application: F (1, 16) = 44.85, P<0.01 and interaction: F (7, 112) = 3.540, P<0.01. Compared with the SNL group, Noggin application provided marked pain relief at day 3, day 4, day 5 and day 7 after SNL. n=9 at each time point for both groups. ( B and C ) Representative Western blotting showed SNL induced CD11b and CD16 upregulation at the first week, while ARG-1 expression remained unchanged compared with the Sham group; Moreover, Noggin effectively decreased the CD11b expressions at P1 and P4 and the CD16 expressions consecutively for the whole week. Simultaneously, the ARG-1 levels significantly elevated from P1 to P7 after Noggin treatment. n=3 for each column. Two-way ANOVA, followed by a Bonferroni test (A) and one-way ANOVA, followed by Sidak’s multiple comparisons test (B and C) were performed to analyze the statistical differences. **Represented P<0.01 compared with the Sham group, # Represented P<0.05 and ## Represented P<0.01 compared with the SNL group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Noggin decreased M1-gene levels and induced a late-stage elevation of M2-gene levels after SNL. The mRNA levels of M1 and M2 subtype were determined by the RT-PCR method. The mRNA relative expressions from the Sham group were set as reference (equal to 1.0). As for the M1 genes, a significant increase in gene expressions were detected in CD16 from P1 to P7 and TNF-α from P4 to P7 in the SNL group compared with the Sham group, except for the MHC-II, which stayed unchanged. Then, in the SNL+NOG group, Noggin application markedly decreased CD16 and TNF-α levels for the 1st week. As for the M2 genes, SNL induced statistical elevation of ARG-1 from P4 and P7 and CD204 from P1 to P7, while IL-4 stayed comparable with the Sham group. Then, in the SNL+NOG group, Noggin treatment prominently increased ARG-1 at P7, CD204 at P4 and P7 and IL-4 at P7. n=3 for each time point. One-way ANOVA, followed by Sidak’s multiple comparisons test were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group; # Represented P<0.05 and ## Represented P<0.01 compared with the SNL group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Reverse Transcription Polymerase Chain Reaction