Journal: Journal of neuroinflammation

Article Title: Microglia induce the transformation of A1/A2 reactive astrocytes via the CXCR7/PI3K/Akt pathway in chronic post-surgical pain.

doi: 10.1186/s12974-020-01891-5

Figure Lengend Snippet: Fig. 1 Experimental designs and animal groups. Experiment 1: changes in pain behavior and glial phenotypes after SMIR in rats. Experiment 2: effects of minocycline pretreatment on mechanical allodynia, glial phenotypes, and the CXCR7/PI3K/Akt pathway after SMIR in rats. Experiment 3: effects of CXCR7 agonist pretreatment on mechanical allodynia after SMIR in rats. Experiment 4: effects of PI3K/Akt pathway inhibitor pretreatment on the analgesic effect of minocycline and AMD3100. WB: western blot; IF: immunofluorescence; ELISA: enzyme-linked immunosorbent assay; SMIR: skin/muscle incision and retraction; AMD3100: CXCR7 agonist; minocycline: microglia inhibitor; LY294002: PI3K inhibitor; PWT: paw withdrawal threshold

Article Snippet: The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (TBST, 0.1%) for 2 h at room temperature, followed by overnight incubation at 4 °C with specific primary antibodies: rabbit anti-C3/C3a antibody (A13283, 1:1000, Abclonal, Wuhan, China), rabbit antiS100A10 antibody (ab187201, 1:500, Abcam, MA, USA), mouse anti-glial fibrillary acidic protein antibody (GFAP, #3670, 1:5000, Cell Signaling Technology, MA, USA), rabbit anti-CXCR7 antibody (ab72100, 1:1000, Abcam), rabbit anti-p-PI3K antibody (AF3241, 1:1000, Affinity, Wuhan, China), mouse anti-PI3K antibody (60225-1-Ig, 1: 1000, Proteintech), mouse anti-p-Akt antibody (66444-1-Ig, 1:1000, Proteintech), rabbit anti-Akt antibody (10176-2-AP, 1:1000, Proteintech), and mouse anti-glyceraldehyde 3- phosphate dehydrogenase (GAPDH) antibody (AC002, 1: 5000, Abclonal).

Techniques: Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroinflammation

Article Title: Microglia induce the transformation of A1/A2 reactive astrocytes via the CXCR7/PI3K/Akt pathway in chronic post-surgical pain.

doi: 10.1186/s12974-020-01891-5

Figure Lengend Snippet: Fig. 5 The CXCR7 and PI3K/Akt signaling pathways are involved in CPSP in the dorsal horn of the spinal cord. a, b, d, e Representative western blot images and quantification of CXCL12 (a), CXCR7 (b), p-PI3K (d), and p-Akt (e) in the spinal cords of sham and SMIR group rats (n = 4). c Double immunofluorescence staining for CXCR7 (red) labeling with GFAP (green) for astrocytes, Iba1 (green) for microglia, or NeuN (green) for neurons in sham and SMIR rats at day 14 after surgery (n = 3). Scale bar: 50 μm or 200 μm. f–h Representative western blot images and quantification of CXCR7, p-PI3K, and p-Akt in the spinal cords of SMIR rats after injection of vehicle or minocycline (n = 3–4). Compared with the sham rats, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The expression of CXCR7 was normalized to GAPDH for each sample, and p-P13K and p-Akt were normalized to PI3K and Akt, respectively, for each sample. The fold change of CXCR7, p-PI3K, and p-Akt in the sham group was set as 1 for quantification. d: day; GFAP: glial fibrillary acidic protein; IBA1: ionized calcium-binding adapter molecule 1; NeuN: neuronal nuclei; SMIR: skin/muscle incision and retraction

Article Snippet: The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (TBST, 0.1%) for 2 h at room temperature, followed by overnight incubation at 4 °C with specific primary antibodies: rabbit anti-C3/C3a antibody (A13283, 1:1000, Abclonal, Wuhan, China), rabbit antiS100A10 antibody (ab187201, 1:500, Abcam, MA, USA), mouse anti-glial fibrillary acidic protein antibody (GFAP, #3670, 1:5000, Cell Signaling Technology, MA, USA), rabbit anti-CXCR7 antibody (ab72100, 1:1000, Abcam), rabbit anti-p-PI3K antibody (AF3241, 1:1000, Affinity, Wuhan, China), mouse anti-PI3K antibody (60225-1-Ig, 1: 1000, Proteintech), mouse anti-p-Akt antibody (66444-1-Ig, 1:1000, Proteintech), rabbit anti-Akt antibody (10176-2-AP, 1:1000, Proteintech), and mouse anti-glyceraldehyde 3- phosphate dehydrogenase (GAPDH) antibody (AC002, 1: 5000, Abclonal).

Techniques: Protein-Protein interactions, Western Blot, Double Immunofluorescence Staining, Labeling, Injection, Expressing, Binding Assay

Journal: Journal of neuroinflammation

Article Title: Microglia induce the transformation of A1/A2 reactive astrocytes via the CXCR7/PI3K/Akt pathway in chronic post-surgical pain.

doi: 10.1186/s12974-020-01891-5

Figure Lengend Snippet: Fig. 6 A CXCR7 agonist provided similar effects to minocycline after SMIR, but there was no synergistic effect with minocycline. AMD3100 alone or combined with minocycline was intrathecally injected immediately and for seven consecutive days after SMIR. a Mechanical allodynia was evaluated by paw withdraw threshold tests (n = 6). b–f Representative western blot images and quantification of CXCR7, C3, S100A10, p-PI3K, and p-Akt in the spinal cords of animals from different groups (n = 4–5). Compared with the SMIR+vehicle group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared with the SMIR+AMD3100+minocycline group, #P < 0.05, ##P < 0.01, ####P < 0.0001. A: AMD3100, a specific agonist of CXCR7; M: minocycline, an inhibitor of microglia; AM: AMD3100+minocycline; BL: baseline; d: day; ipsi: ipsilateral; SMIR: skin/muscle incision and retraction

Article Snippet: The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (TBST, 0.1%) for 2 h at room temperature, followed by overnight incubation at 4 °C with specific primary antibodies: rabbit anti-C3/C3a antibody (A13283, 1:1000, Abclonal, Wuhan, China), rabbit antiS100A10 antibody (ab187201, 1:500, Abcam, MA, USA), mouse anti-glial fibrillary acidic protein antibody (GFAP, #3670, 1:5000, Cell Signaling Technology, MA, USA), rabbit anti-CXCR7 antibody (ab72100, 1:1000, Abcam), rabbit anti-p-PI3K antibody (AF3241, 1:1000, Affinity, Wuhan, China), mouse anti-PI3K antibody (60225-1-Ig, 1: 1000, Proteintech), mouse anti-p-Akt antibody (66444-1-Ig, 1:1000, Proteintech), rabbit anti-Akt antibody (10176-2-AP, 1:1000, Proteintech), and mouse anti-glyceraldehyde 3- phosphate dehydrogenase (GAPDH) antibody (AC002, 1: 5000, Abclonal).

Techniques: Injection, Western Blot

Journal: Journal of neuroinflammation

Article Title: Microglia induce the transformation of A1/A2 reactive astrocytes via the CXCR7/PI3K/Akt pathway in chronic post-surgical pain.

doi: 10.1186/s12974-020-01891-5

Figure Lengend Snippet: Fig. 7 Minocycline and AMD3100 reverted the A1/A2 ratio of reactive astrocytes and improved behavior via PI3K/Akt pathway activation after SMIR. AMD3100 or minocycline was intrathecally injected immediately and for seven consecutive days after SMIR. Rats were administered intrathecal LY294002 before minocycline or AMD3100 administration. a Mechanical allodynia was evaluated by paw withdraw threshold tests (n = 6). b, c Representative western blot images and quantification of C3 and S100A10 in the spinal cords of animals from different groups (n = 5). Compared with the SMIR+vehicle group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared with the SMIR+AMD3100 group or the SMIR+minocycline group, #P < 0.05, ###P < 0.001, ####P < 0.0001. A: AMD3100, a specific agonist of CXCR7; M: minocycline, an inhibitor of microglia; L: LY294002, a specific inhibitor of PI3K; LA: LY294002+AMD3100; LM: LY294002+minocycline; BL: baseline; d: day; ipsi: ipsilateral; SMIR: skin/muscle incision and retraction

Article Snippet: The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (TBST, 0.1%) for 2 h at room temperature, followed by overnight incubation at 4 °C with specific primary antibodies: rabbit anti-C3/C3a antibody (A13283, 1:1000, Abclonal, Wuhan, China), rabbit antiS100A10 antibody (ab187201, 1:500, Abcam, MA, USA), mouse anti-glial fibrillary acidic protein antibody (GFAP, #3670, 1:5000, Cell Signaling Technology, MA, USA), rabbit anti-CXCR7 antibody (ab72100, 1:1000, Abcam), rabbit anti-p-PI3K antibody (AF3241, 1:1000, Affinity, Wuhan, China), mouse anti-PI3K antibody (60225-1-Ig, 1: 1000, Proteintech), mouse anti-p-Akt antibody (66444-1-Ig, 1:1000, Proteintech), rabbit anti-Akt antibody (10176-2-AP, 1:1000, Proteintech), and mouse anti-glyceraldehyde 3- phosphate dehydrogenase (GAPDH) antibody (AC002, 1: 5000, Abclonal).

Techniques: Activation Assay, Injection, Western Blot

Journal: Journal of neuroinflammation

Article Title: Microglia induce the transformation of A1/A2 reactive astrocytes via the CXCR7/PI3K/Akt pathway in chronic post-surgical pain.

doi: 10.1186/s12974-020-01891-5

Figure Lengend Snippet: Fig. 8 Schematic diagram demonstrating that microglia induce the transformation of A1/A2 reactive astrocytes via the CXCR7/PI3K/Akt pathway. During the development of CPSP, microglia are activated first and secrete cytokines such as IL-1α, TNF-α, and C1q, which leads to the downregulation of the CXCR7 receptor and the PI3K/Akt signaling pathway and activate astrocytes, inducing an increase in A1 astrocytes and a decrease in A2 astrocytes. There are other receptors and signaling pathways related to the imbalance in the polarization of astrocytes toward the A1 phenotype during chronic pain, which requires further study

Article Snippet: The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (TBST, 0.1%) for 2 h at room temperature, followed by overnight incubation at 4 °C with specific primary antibodies: rabbit anti-C3/C3a antibody (A13283, 1:1000, Abclonal, Wuhan, China), rabbit antiS100A10 antibody (ab187201, 1:500, Abcam, MA, USA), mouse anti-glial fibrillary acidic protein antibody (GFAP, #3670, 1:5000, Cell Signaling Technology, MA, USA), rabbit anti-CXCR7 antibody (ab72100, 1:1000, Abcam), rabbit anti-p-PI3K antibody (AF3241, 1:1000, Affinity, Wuhan, China), mouse anti-PI3K antibody (60225-1-Ig, 1: 1000, Proteintech), mouse anti-p-Akt antibody (66444-1-Ig, 1:1000, Proteintech), rabbit anti-Akt antibody (10176-2-AP, 1:1000, Proteintech), and mouse anti-glyceraldehyde 3- phosphate dehydrogenase (GAPDH) antibody (AC002, 1: 5000, Abclonal).

Techniques: Transformation Assay, Protein-Protein interactions

Journal: Endocrinology

Article Title: Estrogen receptor-α variant, ER-α36, is involved in tamoxifen resistance and estrogen hypersensitivity.

doi: 10.1210/en.2013-1116

Figure Lengend Snippet: Figure 2. TAM-resistant ER-positive breast cancer MCF7 cells express high concentrations of endogenous ER-36. (A) ER-positive breast cancer MCF7 cells and TAM-resistant MCF7 cells (MCF7/TAM) cells were treated with indicated concentrations of TAM for 7 days, and surviving cells were counted. The columns represent the means of 3 experiments; bars, SE. *P .05 for MCF/TAM cells treated with vehicle vs cells treated with 1M TAM. #P .01 for MCF/TAM cells vs MCF cells treated with indicated concentrations of TAM. (B) Western blot analysis of the expression concentrations of ER-36 and ER-66, EGFR, and HER2 in MCF7 and MCF7/TAM cells.

Article Snippet: Anti-phospho-p44/42 ERK (Thr202/Tyr204) (197G2) mouse monoclonal antibody (mAb), anti-p44/42 ERK (137F5) rabbit mAb, anti-phospho-AKT (Ser473) (D9E) rabbit mAb and anti-AKT (pan) (C67E7) rabbit mAb, anti epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts).

Techniques: Western Blot, Expressing

Journal: Endocrinology

Article Title: Estrogen receptor-α variant, ER-α36, is involved in tamoxifen resistance and estrogen hypersensitivity.

doi: 10.1210/en.2013-1116

Figure Lengend Snippet: Figure 3. ER-36 is involved in TAM resistance. (A) Western blot analysis of the concentrations of ER-36 and ER-66, EGFR, and HER2 proteins in MCF7 cells, MCF7/TAM cells transfected with the empty expression vector (MCF/TAM/Vector), and MCF7/TAM cells transfected with an ER-36-specific shRNA expression vector (MCF7/TAM/Si36). (B) Cells were treated with indicated concentrations of TAM for 7 days, and the numbers of surviving cells were determined. The columns represent the means of 3 experiments; bars, SE. *P .05 for MCF/ TAM/Vector cells treated with vehicle vs cells treated with 1M TAM.

Article Snippet: Anti-phospho-p44/42 ERK (Thr202/Tyr204) (197G2) mouse monoclonal antibody (mAb), anti-p44/42 ERK (137F5) rabbit mAb, anti-phospho-AKT (Ser473) (D9E) rabbit mAb and anti-AKT (pan) (C67E7) rabbit mAb, anti epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, shRNA

Journal: Endocrinology

Article Title: Estrogen receptor-α variant, ER-α36, is involved in tamoxifen resistance and estrogen hypersensitivity.

doi: 10.1210/en.2013-1116

Figure Lengend Snippet: Figure 4. ER-positive breast cancer cells with elevated concentrations of ER-36 protein are resistant to TAM. (A) Western blot analysis of the lysates from ER-positive breast cancer MCF7 cells, MCF7 cells with forced expression of ER-36 (MCF7/ER36), and H3396 cells with high concentrations of endogenous ER-36 protein. The concentrations of EGFR and HER2 proteins are also shown. (B) Cells were treated with indicated concentrations of TAM for 7 days, and the numbers of surviving cells were counted. The columns represent the means of 3 experiments; bars, SE.

Article Snippet: Anti-phospho-p44/42 ERK (Thr202/Tyr204) (197G2) mouse monoclonal antibody (mAb), anti-p44/42 ERK (137F5) rabbit mAb, anti-phospho-AKT (Ser473) (D9E) rabbit mAb and anti-AKT (pan) (C67E7) rabbit mAb, anti epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts).

Techniques: Western Blot, Expressing