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

Article Title: Targeting the phosphoinositide 3-kinase p110-α isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer.

doi: 10.1158/1078-0432.CCR-12-1138

Figure Lengend Snippet: Figure 1. Expression of the PI3K p110-a and p110-b, and the antiapoptotic protein Bcl-2, in SCLC patient samples. A, immunostaining with PI3K p110-a and p110-b showing negative SCLC staining and representative p110-a and p110-b SCLC positive staining, compared with normal lung tissue. B, analysis of the IHC staining PI3K p110-a and p110-b in SCLC patient samples. C, immunostaining with Bcl-2 showing negative and representative Bcl-2 intermediate and strongly positive SCLC staining. D, the mean IHC staining scores Bcl-2 in 20 paraffin-embedded specimens each of normal lung tissue and of 40 SCLC paraffin-embedded specimens. IHC scores ¼ percentage of positive cells staining intensity (for details see Materials and Methods).

Article Snippet: Immunoreactivity was evaluated on commercial tissue microarray (TMA) sections of SCLC [Biomax LC10010; 2 cores: female 9 (22.5%), female age 32–66 years (mean value 52.5 years); male 31 (77.5%), male age 34–76 years (mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7, stage IIIb 2; node-negative 12, node-positive 28 (22N1 and 6 N2)] using the PI3K p110-a (Cell Signaling Technology, 4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signaling) antibodies in a modification of the antigen retrieval technique (13).

Techniques: Expressing, Immunostaining, Staining, Immunohistochemistry

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

Article Title: Targeting the phosphoinositide 3-kinase p110-α isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer.

doi: 10.1158/1078-0432.CCR-12-1138

Figure Lengend Snippet: Figure 2. PI3K inhibition blocks cell viability and PI3K downstream signaling. A and B, the SCLC cell lines H69, H209, H510, and SW2 were incubated with increasing concentrations of the class I PI3K inhibitors PIK75, YM024 (A), TGX221, and IC87114 (B) in serum- containing medium. Cell viability was assessed using the MTS assay after 3 days. The data are mean with SD from 4 replicates and at least 3 independent experiments. C, H69 cells were incubated with increasing concentrations of the PI3K p110-a inhibitors PIK75, YM024, and PI103 (PI3K p110- a/mTOR inhibitor) and the PI3K p110-b inhibitor TGX221. After 24 hours, the cells were harvested and whole-cell lysates analyzed by SDS-PAGE and Western blotting for the proteins indicated.

Article Snippet: Immunoreactivity was evaluated on commercial tissue microarray (TMA) sections of SCLC [Biomax LC10010; 2 cores: female 9 (22.5%), female age 32–66 years (mean value 52.5 years); male 31 (77.5%), male age 34–76 years (mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7, stage IIIb 2; node-negative 12, node-positive 28 (22N1 and 6 N2)] using the PI3K p110-a (Cell Signaling Technology, 4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signaling) antibodies in a modification of the antigen retrieval technique (13).

Techniques: Inhibition, Incubation, MTS Assay, SDS Page, Western Blot

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

Article Title: Targeting the phosphoinositide 3-kinase p110-α isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer.

doi: 10.1158/1078-0432.CCR-12-1138

Figure Lengend Snippet: Figure 3. Silencing of p110-a but not p110-b affects cell viability and PI3K downstream signaling activation. A and C, H69 cells were transiently transfected with siRNA constructs targeting p110-a or p110- b, or nontargeting scrambled control (A) or p110-a was stably silenced by the lentiviral delivery of shRNA constructs (C). Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies for the proteins indicated. B and D, cell viability of H69 cells transiently (B) or stably (D) transfected with constructs targeting p110-a or p110-b was assessed using the MTS assay after 2 and 3 days. A nontargeting construct was used as control.

Article Snippet: Immunoreactivity was evaluated on commercial tissue microarray (TMA) sections of SCLC [Biomax LC10010; 2 cores: female 9 (22.5%), female age 32–66 years (mean value 52.5 years); male 31 (77.5%), male age 34–76 years (mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7, stage IIIb 2; node-negative 12, node-positive 28 (22N1 and 6 N2)] using the PI3K p110-a (Cell Signaling Technology, 4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signaling) antibodies in a modification of the antigen retrieval technique (13).

Techniques: Activation Assay, Transfection, Construct, Control, Stable Transfection, shRNA, SDS Page, Western Blot, MTS Assay

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

Article Title: Targeting the phosphoinositide 3-kinase p110-α isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer.

doi: 10.1158/1078-0432.CCR-12-1138

Figure Lengend Snippet: Figure 4. PI3K inhibition increases apoptosis and autophagy in SCLC. A and C, H69, H209, H510, and SW2 cells grown in serum- containing medium were incubated with increasing concentrations of the PI3K p110-a inhibitors PIK75 and YM024, the PI3K p110-a inhibitor TGX221, PI103 (PI3K p110-a/mTOR inhibitor), RAD001, or etoposide/cisplatin. After 24 hours, the cells were harvested and cell lysates analyzed by SDS- PAGE and Western blotting for the proteins indicated. B and D, H69 cells grown in serum-containing medium were incubated with the PI3K inhibitors PIK75 (0.05 mmol/L) and TGX221 (10 mmol/L), rapamycin (0.1 mg/mL), or etoposide (10 mmol/L) in absence or presence of zVAD-FMK (50 mmol/L) or chloroquine (20 mmol/L). Cell proliferation was assessed using the MTS assay after 12 hours. The data are mean of 4 replicates and 3 independently carried out experiments. , P < 0.01; , P < 0.05.

Article Snippet: Immunoreactivity was evaluated on commercial tissue microarray (TMA) sections of SCLC [Biomax LC10010; 2 cores: female 9 (22.5%), female age 32–66 years (mean value 52.5 years); male 31 (77.5%), male age 34–76 years (mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7, stage IIIb 2; node-negative 12, node-positive 28 (22N1 and 6 N2)] using the PI3K p110-a (Cell Signaling Technology, 4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signaling) antibodies in a modification of the antigen retrieval technique (13).

Techniques: Inhibition, Incubation, SDS Page, Western Blot, MTS Assay

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

Article Title: Targeting the phosphoinositide 3-kinase p110-α isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer.

doi: 10.1158/1078-0432.CCR-12-1138

Figure Lengend Snippet: Figure 5. PI3K inhibition reduces tumor formation and vascularization in vivo. A, H69 cells were treated with vehicle or different concentrations of PIK75 and applied on the CAM of chick embryos. On day 13, pictures and tumors were taken to analyze tumor size and weight (A) and vessel density (B). B, analysis of the vessel density reduction upon PI3K p110-a inhibition in vehicle- or PIK75-treated H69 tumors. C, immunostaining with Ki67 and cleaved caspase-3 comparing the expression in vehicle- and PIK75-treated SCLC tumor sections. D, analysis of Ki67 or caspase-3 positively stained tumor cells comparing vehicle- and PIK75- treated H69 tumor sections. Positively stained cells (%) ¼ number of positive cells/total number of cells 100. , P < 0.001; , P < 0.01; , P < 0.05.

Article Snippet: Immunoreactivity was evaluated on commercial tissue microarray (TMA) sections of SCLC [Biomax LC10010; 2 cores: female 9 (22.5%), female age 32–66 years (mean value 52.5 years); male 31 (77.5%), male age 34–76 years (mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7, stage IIIb 2; node-negative 12, node-positive 28 (22N1 and 6 N2)] using the PI3K p110-a (Cell Signaling Technology, 4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signaling) antibodies in a modification of the antigen retrieval technique (13).

Techniques: Inhibition, In Vivo, Immunostaining, Expressing, Staining

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

Article Title: Targeting the phosphoinositide 3-kinase p110-α isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer.

doi: 10.1158/1078-0432.CCR-12-1138

Figure Lengend Snippet: Figure 6. The proteins of the Bcl-2 family are downstream targets of p110-a. A, gene expression analysis by DNA microarray: heatmap of gene expression changes caused by p110-a inhibition. B–D, H69, H209, and SW2 (B) or H69 (C and D) cells grown in serum-containing medium were incubated with increasing concentrations of the PI3K p110-a inhibitors PIK75 and YM024, the PI3K p110-b inhibitor TGX221, RAD001, and etoposide/ cisplatin or the IKK inhibitor wedelolactone (D). After 24 hours, the cells were harvested and cell lysates analyzed by SDS-PAGE and Western blotting for the proteins indicated.

Article Snippet: Immunoreactivity was evaluated on commercial tissue microarray (TMA) sections of SCLC [Biomax LC10010; 2 cores: female 9 (22.5%), female age 32–66 years (mean value 52.5 years); male 31 (77.5%), male age 34–76 years (mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7, stage IIIb 2; node-negative 12, node-positive 28 (22N1 and 6 N2)] using the PI3K p110-a (Cell Signaling Technology, 4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signaling) antibodies in a modification of the antigen retrieval technique (13).

Techniques: Gene Expression, Microarray, Inhibition, Incubation, SDS Page, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Chronic cerebral hypoperfusion causes decrease of O-GlcNAcylation, hyperphosphorylation of tau and behavioral deficits in mice

doi: 10.3389/fnagi.2014.00010

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: PI3K p85 , Poly- , PI3K (p85) , , Cell Signaling Technology.

Techniques:

Journal: Frontiers in cellular and infection microbiology

Article Title: Streptococcus pneumoniae Endopeptidase O (PepO) Elicits a Strong Innate Immune Response in Mice via TLR2 and TLR4 Signaling Pathways.

doi: 10.3389/fcimb.2016.00023

Figure Lengend Snippet: FIGURE 8 | rPepO-induced cytokines production depends primarily on the activation of p38, Akt and NF-κB. (A–D) PEMs were pretreated with SP600125, U0126, SB203508, LY294002, AG490, and BAY11-7082 for 60 min, and incubated with rPepO (10 µg /ml) for another 24 hr. Cytokines production (TNF-α, IL-6, CXCL1, and CXCL10) were measured by ELISA. The data are shown as the mean ± SD (n = 3). Student’s t-test was performed to calculate the statistical significance (**p < 0.01; ***p < 0.001).

Article Snippet: P38 inhibitor SB203580, ERK inhibitor U0126, JNK inhibitor SP600125, IκB-α phosphorylation inhibitor BAY11-7082, Janus kinase inhibitor AG490,and PI3K inhibitor LY294002 were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay