Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Reagents used for in vitro experiments.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Reagents used for in vitro experiments.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: Extracellular CIRP dysregulates macrophage bacterial phagocytosis in sepsis

doi: 10.1038/s41423-022-00961-3

Figure Lengend Snippet: eCIRP downregulates actin-binding protein expression. RAW 264.7 cells were incubated with rmCIRP at doses of 0.1, 0.2, and 1 µg/ml for 24 h ( A , C , E ) or at a dose of 1 µg/ml for 1, 5 and 24 h ( B , D , F ). ARP2 and p-cofilin protein expression was assessed by Western blotting. A , B Representative blots and C – F the corresponding bar diagram are shown. The experiment was performed three times. The data presented were obtained from three independent experiments and are expressed as the mean ± SD ( n = 6–9/group). The PBS control was set to 1 for normalization. The groups were compared by one-way ANOVA and the SNK method. * p < 0.05 vs. the PBS-treated group. G RAW 264.7 cells were incubated in 1 µg/ml rmCIRP or PBS for 24 h. The cells were examined under a Nikon microscope, and phase-contrast images were acquired. Scale bar, 100 µm

Article Snippet: The antibodies for Western blotting included anti-ARP2 (catalog no: 5614), anti-p-cofilin (catalog no: 3313), anti-total cofilin (catalog no: 5175), anti-βPIX (catalog no: 4515), anti-mouse p-STAT3 (Tyr705, catalog no: 9131), and anti-total STAT3 (catalog no: 9139); these were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Binding Assay, Expressing, Incubation, Western Blot, Microscopy

Journal: Cellular and Molecular Immunology

Article Title: Extracellular CIRP dysregulates macrophage bacterial phagocytosis in sepsis

doi: 10.1038/s41423-022-00961-3

Figure Lengend Snippet: STAT3 activation is crucial to the eCIRP-induced dysregulation of actin remodeling. RAW 264.7 cells were incubated with rmCIRP (1 µg/ml) for 24 h with or without stattic (3 µM). PBS was used as a control. A – C After treatment, the levels of p-STAT3 and ARP2 were evaluated by Western blotting. Data were obtained from two independent experiments and are expressed as the mean ± SD ( n = 8–12/group). The PBS control was set to 1 for normalization. The groups were compared by one-way ANOVA and the SNK method. * p < 0.05 vs. PBS; # p < 0.05 vs. rmCIRP. D , E After treatment, phagocytosis was evaluated by adding pHrodo-labeled E. coli , and microscopy images were acquired at the end of the assay. Representative images are shown. Data were obtained from two independent experiments and are expressed as the mean ± SD ( n = 9–11/group). The experiments were repeated five times. The PBS control was set to 100% for normalization. The groups were compared by one-way ANOVA and the SNK method. * p < 0.05 vs. PBS. # p < 0.05 vs. rmCIRP. F After treatment, the efficiency of phagocytosis was recorded, and the kinetics with 10-min intervals are presented. At the end of the assay, data were analyzed and are expressed as the mean ± SD ( n = 4/group). The groups were compared by one-way ANOVA and the SNK method. * p < 0.05 vs. PBS. # p < 0.05 vs. rmCIR P . G After treatment, cells were fixed, and F-actin was stained with phalloidin. Representative confocal microscopy images are presented. Scale bar, 50 µm. H , I After treatment, the levels of p-cofilin were evaluated by Western blotting. Representative blots are shown. The experiments were repeated three times. Data were obtained from two independent experiments and are expressed as the mean ± SD ( n = 6–8/group). The PBS control was set to 1 for normalization. The groups were compared by one-way ANOVA and the SNK method. * p < 0.05 vs. PBS control; # p < 0.05 vs. rmCIRP. J P-cofilin immunostaining. P-cofilin (red), F-actin (green), and the merged staining images are shown. Scale bar, 50 µm

Article Snippet: The antibodies for Western blotting included anti-ARP2 (catalog no: 5614), anti-p-cofilin (catalog no: 3313), anti-total cofilin (catalog no: 5175), anti-βPIX (catalog no: 4515), anti-mouse p-STAT3 (Tyr705, catalog no: 9131), and anti-total STAT3 (catalog no: 9139); these were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Activation Assay, Incubation, Western Blot, Labeling, Microscopy, Staining, Confocal Microscopy, Immunostaining

Journal: Cellular and Molecular Immunology

Article Title: Extracellular CIRP dysregulates macrophage bacterial phagocytosis in sepsis

doi: 10.1038/s41423-022-00961-3

Figure Lengend Snippet: Graphical summary. Under normal conditions, invading pathogens are detected by surface receptors, such as FcγRs on macrophages, and initiate actin remodeling by activating Rac1 to regulate ARP2 and cofilin activities. βPIX regulates Rac1 from the inactive GDP-bound state (GDP-Rac1) to the activated GTP-bound state (GTP-Rac1). Sepsis causes increased release of eCIRP. eCIRP-induced STAT3 phosphorylation causes STAT3 and βPIX to form a complex. Thus, Rac1 activation is inhibited, and actin remodeling is interrupted, resulting in an impairment in the phagocytosis of bacteria

Article Snippet: The antibodies for Western blotting included anti-ARP2 (catalog no: 5614), anti-p-cofilin (catalog no: 3313), anti-total cofilin (catalog no: 5175), anti-βPIX (catalog no: 4515), anti-mouse p-STAT3 (Tyr705, catalog no: 9131), and anti-total STAT3 (catalog no: 9139); these were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Activation Assay

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Arp2 expression in BUC specimens vs paired normal bladder tissue, and positive lymph node specimens

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Association of Arp2 expression with clinicopathological features of bladder urothelial carcinoma patients

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Association of clinicopathological features with lymph node metastasis of bladder urothelial carcinoma

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Multivariate analysis of clinicopathological features for association with lymph node metastasis of BUC

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques:

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Univariate and multivariate Cox proportional hazard analyses for the 5-year recurrence-free survival

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques:

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Univariate and multivariate Cox proportional hazard analyses for the 5-year overall survival

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques:

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Tissue microarrays containing BUC and normal bladder tissues were immunostained with a monoclonal anti-Arp2 antibody, and the data were semiquantitatively analyzed: ( A ) positive lymph nodes; ( B ) BUC; and ( C ) normal bladder tissue; 200 ¥ magnification. Note: The expression of Arp2 in the positive lymph nodes was higher than that in the paired BUC, and the expression of Arp2 in the BUC was higher than that in the paired normal bladder tissue. Abbreviations: Arp2, actin-related protein 2/3 complex subunit 2; BUC, bladder urothelial carcinoma.

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Different expression levels of Arp2 protein in BUC (400 ¥ magnification). Notes: ( A ) Negative (score of 0), ( B ) weakly positive (score of 2), ( C ) positive (score of 5), and ( D ) strongly positive (score of 7) Arp2 protein expression. Abbreviations: Arp2, actin-related protein 2/3 complex subunit 2; BUC, bladder urothelial carcinoma.

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Kaplan–Meier curve analysis of the 5-year RFS of BUC patients stratified by Arp2 expression. Note: Increased Arp2 expression was associated with a shortened 5-year RFS (log-rank test, P < 0.001). Abbreviations: Arp2, actin-related protein 2/3 complex subunit 2; BUC, bladder urothelial carcinoma; RFS, recurrence-free survival.

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma

doi: 10.2147/CMAR.S155453

Figure Lengend Snippet: Kaplan–Meier curves of the 5-year OS of BUC patients stratified by Arp2 expression. Note: Increased Arp2 expression was associated with a shortened 5-year OS (log-rank test, P < 0.001). Abbreviations: Arp2, actin-related protein 2/3 complex subunit 2; BUC, bladder urothelial carcinoma; OS, overall survival.

Article Snippet: After that, to block endogenous peroxidase activity, the TMAs were then incubated with 3% H 2 O 2 for 10 min followed by 20% normal goat serum at room temperature for 30 min. Microarray sections were incubated with a rabbit monoclonal anti-Arp2 antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:400 at 4°C overnight.

Techniques: Expressing