Journal: Molecular human reproduction

Article Title: IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling.

doi: 10.1093/molehr/gat094

Figure Lengend Snippet: Figure 4 Effects of IL-33/ST2L/sST2 axis on CCL2/CCR2 expres- sion in DSCs. The secretion of chemokine CCL2 (A) by DSCs was determined by ELISA, and the expression of CCR2 on DSCs (B) by flow cytometry. DSCs (2 × 105 cells/well) were seeded in 24-well plates with or without rhIL-33(0.01–10 ng/ml) and sST2 (100– 400 ng/ml). The supernatant was then collected after 48 h while DSCs were used to detect the protein concentration, and the CCL2 levelofeachgroupwascalculatedastheratiooftheCCL2concentration of supernatant to the protein concentration. The pictures are from a representative experiment and the numbers are the percentages of positive cells. Results are representative of three independent experi- ments. ***P , 0.001. CCL2-APC, allophycocyanin labeled CCL2 antibody.

Article Snippet: BrdU cell proliferation assay Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) cell proliferation assay was applied to evaluate the effect of IL-33 on cell proliferation with or without sST2, anti-CCL2 neutralizing antibody (MAB279, R&D Systems) and CCR2 blocker RS102895 (R1903, Sigma-Aldrich, Santa Clara, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Cytometry, Protein Concentration, Labeling

Journal: Molecular human reproduction

Article Title: IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling.

doi: 10.1093/molehr/gat094

Figure Lengend Snippet: Figure 5 The increased proliferation and invasiveness of DSCs induced by IL-33 are dependent on CCL2/CCR2 interaction. DSCs (1 × 104 cell/well, BrdUproliferationassay;3 × 105 cells/well,cellcycleassay;2 × 105 cells/well,invasionassay)weretreatedwithrhIL-33(1 ng/ml),a-CCL2(1 mg/ml),or CCR2 blocker RS102895(100 ng/ml) with vehicle as control. After stimulation with rhIL-33 (1 ng/ml), the cells were treated with CCL2 neutralizing anti- body (1 mg/ml) or RS102895 (100 ng/ml) foranother 48 h. Thereafter, BrdU proliferation assay(A), cell cycle (B) and invasion assay (C) wereconducted to analyze proliferation and invasiveness of DSCs, respectively. Error bars depict the standard deviation of the sample mean. The images are from an ex- periment that is representative of three independently conducted experiments. The x-axis, from the left, represents the cell cycle phases of G1(green region), S(the region between G1 and G2) and G2(blue region). *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: BrdU cell proliferation assay Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) cell proliferation assay was applied to evaluate the effect of IL-33 on cell proliferation with or without sST2, anti-CCL2 neutralizing antibody (MAB279, R&D Systems) and CCR2 blocker RS102895 (R1903, Sigma-Aldrich, Santa Clara, USA).

Techniques: Control, Proliferation Assay, Invasion Assay, Standard Deviation

Journal: Molecular human reproduction

Article Title: IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling.

doi: 10.1093/molehr/gat094

Figure Lengend Snippet: Figure 8 Schematic representation of IL-33 effects on regulating biological behavior of DSCs. IL-33 can stimulate CCL2/CCR2 expression through NF-kB and ERK1/2 signal pathways, thereby enhancing proliferation and invasion of DSCs. At the same time, proliferation relative gene (PCNA, survivin) and invasion relative gene (titin, MMP2) are also increased in response to IL-33, which further promotes prolifertation and invasiveness of DSCs. sST2 can block IL-33/ST2 signaling through bounding to IL-33.

Article Snippet: BrdU cell proliferation assay Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) cell proliferation assay was applied to evaluate the effect of IL-33 on cell proliferation with or without sST2, anti-CCL2 neutralizing antibody (MAB279, R&D Systems) and CCR2 blocker RS102895 (R1903, Sigma-Aldrich, Santa Clara, USA).

Techniques: Expressing, Blocking Assay

Journal: Frontiers in Pharmacology

Article Title: Ningmitai capsules have anti-inflammatory and pain-relieving effects in the chronic prostatitis/chronic pelvic pain syndrome mouse model through systemic immunity

doi: 10.3389/fphar.2022.949316

Figure Lengend Snippet: Antibody information used in the article.

Article Snippet: MCP1/CCL2 (5H2) Mouse mAb , 1:1,000(WB) , ZENBIO(220691).

Techniques:

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 1. Time course of mechanical hypersensitivity induced by paw incision after intrathecal treatment with anti-chemokine (C-C motif) ligand 2 (CCL2) immunoglobulin G (IgG) (3 ng, dotted bars; 10 ng, gray bars) or control IgG (10 ng, open bars) was examined acutely for several minutes after treatment beginning 1 day after surgical incision (A) and for several days after single treatment beginning 1 day after surgical incision (B). Withdrawal thresholds to von Frey filaments values are expressed as the median (solid line) and 25th and 75th percentiles of 8–14 rats per group. * P 0.05 for comparisons to control IgG values at each time point or # P 0.05 for comparison between 3 ng anti-CCL2– and 10 ng anti- CCL2–treated values at each time point by Kruskal-Wallis analysis of variance on ranks followed by Dunn post hoc test. Black ar- rowhead indicates time point of intrathecal injection.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Control, Comparison, Injection

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 2. Representative images of microglial staining with an antibody against ionized calcium-binding adapter molecule 1 (IBA1). Ipsilateral lumbar dorsal spinal cord of rats that underwent sham procedures administered control immunoglobulin G (IgG) (10 ng; A, B, and C) or ipsilateral spinal cord of rats with plantar incision administered 10 ng anti-chemokine (C-C motif) ligand 2 (CCL2) IgG (G, H, and I) or 10 ng control IgG (D, E, and F). Microglial IBA1 immunostaining was increased in the ipsilateral dorsal spinal cord of rats 1 day and 2 days postoperatively (1 dpo and 2 dpo) after plantar incision compared with the levels in rats that underwent sham procedure. A single intrathecal injection of anti-CCL2 IgG (10 ng) administered 1 day after plantar incision reduced the levels of ionized calcium-binding adaptor molecule 1 (IBA1)-immunoreactivity (IR) in the spinal cord of incision rats 24 h after administration (H compared with E) but not 30 min after administration (G compared with D) compared with incision rats that received intrathecal control IgG. Insets depict the morphologic differences in microglia 48 h after incision. Not increased size of microglia in control IgG-treated rats versus anti-CCL2–treated rats (F compared with I). Scale bar in H 150 m and scale bar in I 120 m.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Staining, Binding Assay, Control, Immunostaining, Injection

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 3. Quantification of ionized calcium-binding adapter molecule 1 (IBA1) immunostaining in ipsilateral (ipsi) (gray bar) and contralateral (contra) (open bar) dorsal spinal cord of sham-operated and incision rats 30 min after intrathecal administration of anti-chemokine (C-C motif) ligand 2 (CCL2) (10 ng) or control immunoglobulin G (IgG) (10 ng) on day 1 postoperatively (1 dpo; A) or 24 h after administration of anti-CCL2 (10 ng) or control IgG (10 ng) on day 2 postoperatively (2 dpo; B). * P 0.05 compared with sham control IgG value within side. # P 0.05 compared with contralateral side within treatment group. P 0.05 compared with incision control IgG-treated values by two-way analysis of variance with Bonferroni post hoc test (n 8 rats per group).

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Immunostaining, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 4. Representative images of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) labeling (A, D, and G) and Cd11b (OX42; B, E, and H) in the ipsilateral dorsal spinal cord of sham-operated (A, B, and C) and incision rats treated with 10 ng control immunoglobulin G (IgG) (D, E, and F) or 10 ng anti-chemokine (C-C motif) ligand 2 (CCL2) IgG (G, H, and I) rats 2 days postoperatively (2 dpo) after plantar incision. Rats received a single acute administration of anti-CCL2 IgG (10 ng) on day 1 after plantar incision. High-power confocal images show colocalization of p-p38 MAPK (red) and OX42 (green). Increased levels of cytoplasmic p-p38 MAPK in OX42 immunoreactive microglia of incision rats treated with control IgG compared with sham-operated rats (F compared with C) and incision rats treated with anti-CCL2 IgG (F compared with I). Fluorescent images in panels A, B, D, E, G, and H were inverted in Photoshop (Adobe Systems Inc., San Jose, CA) and converted to grayscale to enhance contrast. Scale bars in G and H 75 m and scale bar in I 5 m.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Labeling, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 5. Quantification of microglial phosphorylated p38 mitogen-ac- tivated protein kinase immunostaining in ipsilateral (ipsi) (gray bar) and contralateral (contra) (open bar) dorsal spinal cord of sham- operated and incision rats 30 min after intrathecal administration of anti-chemokine (C-C motif) ligand 2 (CCL2) (10 ng) or control immu- noglobulin G (IgG) (10 ng) on day 1 postoperatively (1 dpo; A) or 24 h after administration of anti-CCL2 (10 ng) or control IgG (10 ng) on day 2 postoperatively (2 dpo; B). * P 0.05 compared with sham control IgG value within side. # P 0.05 compared with contralateral side within the treatment group. P 0.05 compared with incision control IgG-treated values by two-way analysis of variance with Bon- ferroni post hoc test (n 8 rats per group).

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Immunostaining, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 6. Correlation between spinal markers of microglial activation and behavioral measures of mechanical hypersensitivity 2 days after surgical incision or sham procedure. The association between num- ber of microglial ionized calcium-binding adaptor molecule 1 immu- noreactive (IBA1-IR) pixels (A) or number of microglia positive for phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) (B) and mechanical paw withdrawal thresholds (g) were ex- amined using Spearman rho nonparametric linear regression analysis. CCL2 chemokine (C-C motif) ligand 2; IgG immunoglobulin G.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CPEB3 inhibits epithelial-mesenchymal transition by disrupting the crosstalk between colorectal cancer cells and tumor-associated macrophages via IL-6R/STAT3 signaling

doi: 10.1186/s13046-020-01637-4

Figure Lengend Snippet: CPEB3 modulates CCL2 secretion in CRC cell supernatants to regulate TAM polarization (a) We measured the expression of the respective inflammatory cytokines in cell culture supernatants of HCT116-Ctrl/CPEB3 cells by ProcartaPlex combinable panels; error bars, SEM. (b) We measured the expression of the respective inflammatory cytokines in the supernatants of LoVo-shCtrl/shCPEB3 cells by ProcartaPlex combinable panels; error bars, SEM. (c) THP-1 macrophages were co-cultured with LoVo-shCtrl/shCPEB3 with or without CCL2-neutralizing antibody (1 μg/mL) for 24 h. Flow cytometry was used to explore the surface expression of CD86 and CD163 in the differentiated macrophages; error bars, SEM. (d) THP-1 macrophages were co-cultured with RKO-shCtrl/shCPEB3 cells with or without a CCL2-neutralizing antibody (1 μg/mL) for 24 h. Flow cytometry was used to explore the surface expression of CD86 and CD163 in the differentiated macrophages. Error bars, SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: THP-1 macrophages were co-cultured with stably transfected CRC cells along with or without neutralizing antibodies to CCL2 (anti-CCL2; R&D Systems).

Techniques: Expressing, Cell Culture, Flow Cytometry

Journal: JCI Insight

Article Title: The m 6 A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment

doi: 10.1172/jci.insight.162436

Figure Lengend Snippet: ( A ) Schematic diagram of the MSC and monocyte coculture system. ( B and C ) m 6 A modification levels of MCP1 mRNA of MSCs ( n = 3) cultured without (0 hour) or with monocytes for 6 hours, 12 hours, and 36 hours. ( D and E ) Relative mRNA expression of MCP1 and METTL16 of MSCs ( n = 9) cultured with monocytes at different time points. ( F ) Representative blot images of MCP1, METTL16, METTL14, METTL3, ALKBH5, and FTO of MSCs ( n = 9) cultured with monocytes at different time points. ( G and H ) The mean intensity ratio of MCP1 and METTL16 of MSCs ( n = 9) cultured with monocytes at different time points. ( I ) The correlation between MCP1 mRNA and METTL16 mRNA expression in the MSCs cocultured with monocytes ( R 2 = 0.7760, P < 0.0001). Data are presented as the mean ± SD. One-way ANOVA followed by Bonferroni’s test was performed by comparison with the 0-hour group ( B – E , G , and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. MSCs, mesenchymal stem cells; MCP1, monocyte chemoattractant protein 1.

Article Snippet: Then, the culture supernatant was replaced with FBS-free culture supernatant with or without 0.5 μg/mL anti-MCP1 neutralizing antibody (MAB679, R&D Systems), and 1 × 10 6 monocytes in 100 μL of FBS-culture supernatant were seeded in the upper chambers after staining with CFSE for 15 minutes.

Techniques: Modification, Cell Culture, Expressing, Comparison

Journal: JCI Insight

Article Title: The m 6 A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment

doi: 10.1172/jci.insight.162436

Figure Lengend Snippet: ( A ) Relative MCP1 mRNA expression in siNC- or siMETTL16-treated MSCs ( n = 9) cultured with or without monocytes. ( B ) Representative blot images of MCP1 in siNC- or siMETTL16-treated MSCs ( n = 9) cultured with or without monocytes. ( C ) The mean intensity ratio of MCP1 in siNC- or siMETTL16-treated MSCs ( n = 9) cultured with or without monocytes. ( D ) Relative mRNA expression of MCP1 in OE NC–treated or OE METTL16–treated MSCs ( n = 9) cultured with or without monocytes. ( E ) Representative blot images of MCP1 in OE NC–treated or OE METTL16–treated MSCs ( n = 9) cultured with or without monocytes. ( F ) The mean intensity ratio of MCP1 in OE NC– or OE METTL16–treated MSCs ( n = 9) cultured with or without monocytes. ( G ) Schematic diagram of the monocyte recruitment system. ( H and I ) Representative flow cytometry histograms and relative cell count of monocytes recruited by MSCs ( n = 9). ( J ) Relative cell count of monocytes recruited by siNC- or siMETTL16-treated MSCs ( n = 9) with or without MCP1-neutralizing antibody. Data are presented as the mean ± SD. Two-tailed Student’s t test was performed in panels A , C , D , F , and I and 1-way ANOVA followed by Bonferroni’s test was performed in J . * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. MSCs, mesenchymal stem cells; MCP1, monocyte chemoattractant protein 1; siNC, control siRNA; siMETTL16, siRNA for METTL16; OE NC, control lentiviruses; OE METTL16, lentiviruses overexpressing METTL16.

Article Snippet: Then, the culture supernatant was replaced with FBS-free culture supernatant with or without 0.5 μg/mL anti-MCP1 neutralizing antibody (MAB679, R&D Systems), and 1 × 10 6 monocytes in 100 μL of FBS-culture supernatant were seeded in the upper chambers after staining with CFSE for 15 minutes.

Techniques: Expressing, Cell Culture, Flow Cytometry, Cell Counting, Two Tailed Test, Control

Journal: JCI Insight

Article Title: The m 6 A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment

doi: 10.1172/jci.insight.162436

Figure Lengend Snippet: ( A and B ) m 6 A modification levels of MCP1 mRNA in siNC-/siMETTL16- or OE NC–/OE METTL16–treated MSCs ( n = 3). ( C ) MCP1 promoter activity in siNC- or siMETTL16-treated MSCs ( n = 9) expressed as the ratio of firefly versus Renilla luciferase (Fluc/Rluc). ( D ) Relative expression of MCP1 precursor mRNA (pre-MCP1) and mature mRNA (MCP1) in siNC- or siMETTL16-treated MSCs ( n = 9). ( E ) Degradation curves of MCP1 mRNA in OE NC– or OE METTL16–treated MSCs ( n = 9). ( F ) Degradation curves of MCP1 mRNA in siNC- or siMETTL16-treated MSCs ( n = 9). ( G ) The t 1/2 analysis of MCP1 mRNA in siNC-/siMETTL16- or OE NC–/OE METTL16–treated MSCs ( n = 9). ( H ) The abundance of MCP1 mRNA on polysomes was not changed significantly with knockdown of METTL16 ( n = 9). Data are presented as the mean ± SD. Two-tailed Student’s t test was performed in panels A – D and G . * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS, not significant; MSCs, mesenchymal stem cells. MCP1, monocyte chemoattractant protein 1; siNC, control siRNA; siMETTL16, siRNA for METTL16; OE NC, control lentiviruses; OE METTL16, lentiviruses overexpressing METTL16.

Article Snippet: Then, the culture supernatant was replaced with FBS-free culture supernatant with or without 0.5 μg/mL anti-MCP1 neutralizing antibody (MAB679, R&D Systems), and 1 × 10 6 monocytes in 100 μL of FBS-culture supernatant were seeded in the upper chambers after staining with CFSE for 15 minutes.

Techniques: Modification, Activity Assay, Luciferase, Expressing, Knockdown, Two Tailed Test, Control

Journal: JCI Insight

Article Title: The m 6 A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment

doi: 10.1172/jci.insight.162436

Figure Lengend Snippet: ( A ) Relative MCP1 mRNA expression in siNC-, siYTHDF2-, or siYTHDC2-treated MSCs ( n = 9). ( B ) Representative blot images and mean intensity ratio of MCP1 in siNC-, siYTHDF2-, or siYTHDC2-treated MSCs ( n = 9). ( C and D ) m 6 A modification level of MCP1 mRNA in siNC- or siYTHDF2-treated MSCs ( n = 3). ( E ) Degradation curves of MCP1 mRNA in siNC- or siYTHDF2-treated MSCs ( n = 9). ( F ) The t 1/2 analysis of MCP1 mRNA in siNC- or siYTHDF2-treated MSCs ( n = 9). ( G and H ) YTHDF2 RIP-qPCR analysis of MCP1 mRNA in siNC- or siMETTL16-treated MSCs ( n = 9). Data are presented as the mean ± SD. Two-tailed Student’s t test was performed in panels C , D , G , and H and 1-way ANOVA followed by Bonferroni’s test was performed in A , B , and F . ** P < 0.01; **** P < 0.0001. NS, not significant; MSCs, mesenchymal stem cells; MCP1, monocyte chemoattractant protein 1; siNC, control siRNA; siMETTL16, siRNA for METTL16; siYTHDF2, siRNA for YTHDF2; siYTHDC2, siRNA for YTHDC2.

Article Snippet: Then, the culture supernatant was replaced with FBS-free culture supernatant with or without 0.5 μg/mL anti-MCP1 neutralizing antibody (MAB679, R&D Systems), and 1 × 10 6 monocytes in 100 μL of FBS-culture supernatant were seeded in the upper chambers after staining with CFSE for 15 minutes.

Techniques: Expressing, Modification, Two Tailed Test, Control

Journal: JCI Insight

Article Title: The m 6 A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment

doi: 10.1172/jci.insight.162436

Figure Lengend Snippet: ( A ) Schematic representation of m 6 A sites of MCP1 mRNA in the CDS region. ( B ) Schematic representation of mutated m 6 A sites of MCP1 mRNA in the pCDNA3.1 vector. Adenines in chr17:34256757 (MCP1-Mut1) and chr17:34256822 (MCP1-Mut2) were mutated to guanine. ( C ) pCDNA3.1 vectors expressing the MCP1 mutant were transfected into 293T cells treated with siNC or siYTHDF2, and the relative MCP1 mRNA expression was quantified by qPCR ( n = 9). ( D ) Representative blot images of MCP1 in siNC- or siYTHDF2-treated 293T cells transfected with MCP1-mutant pCDNA3.1 vectors ( n = 9). ( E and F ) YTHDF2 RIP-qPCR analysis of MCP1 mRNA in the MSCs ( n = 9) transfected with MCP1-mutant pCDNA3.1 vectors. Data are presented as the mean ± SD. Two-tailed Student’s t test was performed in panels C and E and 1-way ANOVA followed by Bonferroni’s test was performed in F . * P < 0.05; **** P < 0.0001. NS, not significant; MSCs, mesenchymal stem cells; MCP1, monocyte chemoattractant protein 1; siNC, control siRNA; siYTHDF2, siRNA for YTHDF2.

Article Snippet: Then, the culture supernatant was replaced with FBS-free culture supernatant with or without 0.5 μg/mL anti-MCP1 neutralizing antibody (MAB679, R&D Systems), and 1 × 10 6 monocytes in 100 μL of FBS-culture supernatant were seeded in the upper chambers after staining with CFSE for 15 minutes.

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Transfection, Two Tailed Test, Control