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Servicebio Inc murine pdac cell line panc 02

Mast cell infiltration and degranulation are enhanced in <t>PDAC</t> with abdominal hyperalgesia. (A) Schematic diagram and representative H&E staining showing the boundary between tumor and para-tumor tissues. Para-tumor regions were defined as areas 4 mm away from the tumor margin. The red dashed line indicated the boundary between tumor and adjacent tissue. Scale bar = 2.5 mm. (B) Representative H&E, toluidine blue, and immunofluorescence staining of tumor and para-tumor tissue sections from paired patients with PDAC with or without abdominal pain (n = 10 vs 10). Black arrows indicate toluidine blue + mast cells. White arrows denote tryptase + CD117 + degranulating mast cells. Scale bar = 50 μm. Green, CD117; Red, tryptase; Blue, DAPI. (C and D) Quantification of toluidine blue + mast cells and tryptase + CD117 + degranulating mast cells demonstrated a significant increase in para-tumor tissues from painful patients with PDAC compared with nonpainful patients. Five random fields of view were selected from para-tumor and tumor regions, analyzed using ImageJ, and averaged. (E) ELISA assays revealed significantly elevated levels of histamine (His), tryptase (TPS), and IL-4 in para-tumor tissues from painful patients with PDAC. (F) ELISA of an additional serum cohort (n = 11 vs 11) verified elevated His, TPS, and IL-4 in painful patients with PDAC. (G) Orthotopic PDAC models were established <t>using</t> <t>Panc-02</t> cells. The red dashed line indicated the boundary between tumor and para-tumor tissue in mice, with para-tumor area defined as 3 mm away from the tumor margin. (H) The frequency of withdrawal responses to von Frey filament stimulation was significantly increased in orthotopic PDAC models. (I and J) ELISA assays demonstrated elevated levels of His, TPS, and IL-4 in the serum and para-tumor tissues of PDAC models. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (C–F) or Student t test (H–J). * P < 0.05, ** P < 0.01, *** P < 0.001. DAPI, 4',6-Diamidino-2-Phenylindole; ELISA, enzyme-linked immunosorbent assay; His, histamine; IL-4, interleukin-4; NC, normal control mice subjected to sham surgery; PC, orthotopic pancreatic cancer model mice; PDAC, pancreatic ductal adenocarcinoma; TPS, tryptase.

Murine Pdac Cell Line Panc 02, supplied by Servicebio Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling"

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

Journal: Pain

doi: 10.1097/j.pain.0000000000003909

Mast cell infiltration and degranulation are enhanced in PDAC with abdominal hyperalgesia. (A) Schematic diagram and representative H&E staining showing the boundary between tumor and para-tumor tissues. Para-tumor regions were defined as areas 4 mm away from the tumor margin. The red dashed line indicated the boundary between tumor and adjacent tissue. Scale bar = 2.5 mm. (B) Representative H&E, toluidine blue, and immunofluorescence staining of tumor and para-tumor tissue sections from paired patients with PDAC with or without abdominal pain (n = 10 vs 10). Black arrows indicate toluidine blue + mast cells. White arrows denote tryptase + CD117 + degranulating mast cells. Scale bar = 50 μm. Green, CD117; Red, tryptase; Blue, DAPI. (C and D) Quantification of toluidine blue + mast cells and tryptase + CD117 + degranulating mast cells demonstrated a significant increase in para-tumor tissues from painful patients with PDAC compared with nonpainful patients. Five random fields of view were selected from para-tumor and tumor regions, analyzed using ImageJ, and averaged. (E) ELISA assays revealed significantly elevated levels of histamine (His), tryptase (TPS), and IL-4 in para-tumor tissues from painful patients with PDAC. (F) ELISA of an additional serum cohort (n = 11 vs 11) verified elevated His, TPS, and IL-4 in painful patients with PDAC. (G) Orthotopic PDAC models were established using Panc-02 cells. The red dashed line indicated the boundary between tumor and para-tumor tissue in mice, with para-tumor area defined as 3 mm away from the tumor margin. (H) The frequency of withdrawal responses to von Frey filament stimulation was significantly increased in orthotopic PDAC models. (I and J) ELISA assays demonstrated elevated levels of His, TPS, and IL-4 in the serum and para-tumor tissues of PDAC models. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (C–F) or Student t test (H–J). * P < 0.05, ** P < 0.01, *** P < 0.001. DAPI, 4',6-Diamidino-2-Phenylindole; ELISA, enzyme-linked immunosorbent assay; His, histamine; IL-4, interleukin-4; NC, normal control mice subjected to sham surgery; PC, orthotopic pancreatic cancer model mice; PDAC, pancreatic ductal adenocarcinoma; TPS, tryptase.

Figure Legend Snippet: Mast cell infiltration and degranulation are enhanced in PDAC with abdominal hyperalgesia. (A) Schematic diagram and representative H&E staining showing the boundary between tumor and para-tumor tissues. Para-tumor regions were defined as areas 4 mm away from the tumor margin. The red dashed line indicated the boundary between tumor and adjacent tissue. Scale bar = 2.5 mm. (B) Representative H&E, toluidine blue, and immunofluorescence staining of tumor and para-tumor tissue sections from paired patients with PDAC with or without abdominal pain (n = 10 vs 10). Black arrows indicate toluidine blue + mast cells. White arrows denote tryptase + CD117 + degranulating mast cells. Scale bar = 50 μm. Green, CD117; Red, tryptase; Blue, DAPI. (C and D) Quantification of toluidine blue + mast cells and tryptase + CD117 + degranulating mast cells demonstrated a significant increase in para-tumor tissues from painful patients with PDAC compared with nonpainful patients. Five random fields of view were selected from para-tumor and tumor regions, analyzed using ImageJ, and averaged. (E) ELISA assays revealed significantly elevated levels of histamine (His), tryptase (TPS), and IL-4 in para-tumor tissues from painful patients with PDAC. (F) ELISA of an additional serum cohort (n = 11 vs 11) verified elevated His, TPS, and IL-4 in painful patients with PDAC. (G) Orthotopic PDAC models were established using Panc-02 cells. The red dashed line indicated the boundary between tumor and para-tumor tissue in mice, with para-tumor area defined as 3 mm away from the tumor margin. (H) The frequency of withdrawal responses to von Frey filament stimulation was significantly increased in orthotopic PDAC models. (I and J) ELISA assays demonstrated elevated levels of His, TPS, and IL-4 in the serum and para-tumor tissues of PDAC models. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (C–F) or Student t test (H–J). * P < 0.05, ** P < 0.01, *** P < 0.001. DAPI, 4',6-Diamidino-2-Phenylindole; ELISA, enzyme-linked immunosorbent assay; His, histamine; IL-4, interleukin-4; NC, normal control mice subjected to sham surgery; PC, orthotopic pancreatic cancer model mice; PDAC, pancreatic ductal adenocarcinoma; TPS, tryptase.

Techniques Used: Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Control

Mast cell CRFR1 expression is elevated in patients with PDAC and mouse models. (A and B) CRF levels increased in both patients with PDAC and mouse models with abdominal hyperalgesia based on ELISA assay. (C) UMAP plot of reanalyzed scRNA-seq data from dataset GSE155698 showing distinct pancreatic cell populations. (D) The percentage of mast cell cluster increased in PDAC tissues compared to normal pancreatic tissues. (E) Average CRFR1 expression per mast cell was significantly elevated in PDAC tissues, whereas CRFR2 expression remained unchanged. (F and G) qRT-PCR and western blotting confirmed the upregulation of CRFR1 in orthotopic PDAC mouse models. (H and I) Flow cytometry demonstrated an increased proportion of CD117 + FcεR + mast cells (gated within CD45 + leukocytes) and elevated CRFR1 expression in CD117 + FcεR + CD45 + mast cells in PDAC mice. (J–M) Immunofluorescence staining further confirmed elevated CRFR1 expression in tryptase + mast cells in PDAC experimental mice and painful human patients with PDAC. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (A, M) or Student t test (B, F, I, K). * P < 0.05, ** P < 0.01, *** P < 0.001. CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; UMAP, uniform manifold approximation and projection.

Figure Legend Snippet: Mast cell CRFR1 expression is elevated in patients with PDAC and mouse models. (A and B) CRF levels increased in both patients with PDAC and mouse models with abdominal hyperalgesia based on ELISA assay. (C) UMAP plot of reanalyzed scRNA-seq data from dataset GSE155698 showing distinct pancreatic cell populations. (D) The percentage of mast cell cluster increased in PDAC tissues compared to normal pancreatic tissues. (E) Average CRFR1 expression per mast cell was significantly elevated in PDAC tissues, whereas CRFR2 expression remained unchanged. (F and G) qRT-PCR and western blotting confirmed the upregulation of CRFR1 in orthotopic PDAC mouse models. (H and I) Flow cytometry demonstrated an increased proportion of CD117 + FcεR + mast cells (gated within CD45 + leukocytes) and elevated CRFR1 expression in CD117 + FcεR + CD45 + mast cells in PDAC mice. (J–M) Immunofluorescence staining further confirmed elevated CRFR1 expression in tryptase + mast cells in PDAC experimental mice and painful human patients with PDAC. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (A, M) or Student t test (B, F, I, K). * P < 0.05, ** P < 0.01, *** P < 0.001. CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; UMAP, uniform manifold approximation and projection.

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Immunofluorescence, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction

CRFR1 promotes the activation of mast cell and enhances abdominal pain sensitivity. (A) qRT-PCR verified that CRF (1 μM) significantly upregulated mast cell activation markers, including TPSAB1, IL-4, TNF-α, and CCL2. (B) Western blotting confirmed the increased protein expression of TPSAB1 after CRF stimulation. (C) ELISA assays demonstrated elevated levels of secreted TPS, His, IL-4, TNF-α, and CCL2 in the supernatant of CRF-treated cells. (D) Administration of ANT (a CRFR1 antagonist) significantly reduced abdominal withdrawal response frequency in PDAC mice. (E and F) qRT-PCR and western blotting revealed reduced expression of TPSAB1, IL-4, TNF-α, and CCL2 after ANT treatment. (G) ELISA results further confirmed the suppression of TPS, His, IL-4, TNF-α, and CCL2 secretion in the ANT-treated group. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using 2-tailed Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001. ANT, Antisauvagine; CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor-alpha; TPS, tryptase; TPSAB1, tryptase alpha/beta 1.

Figure Legend Snippet: CRFR1 promotes the activation of mast cell and enhances abdominal pain sensitivity. (A) qRT-PCR verified that CRF (1 μM) significantly upregulated mast cell activation markers, including TPSAB1, IL-4, TNF-α, and CCL2. (B) Western blotting confirmed the increased protein expression of TPSAB1 after CRF stimulation. (C) ELISA assays demonstrated elevated levels of secreted TPS, His, IL-4, TNF-α, and CCL2 in the supernatant of CRF-treated cells. (D) Administration of ANT (a CRFR1 antagonist) significantly reduced abdominal withdrawal response frequency in PDAC mice. (E and F) qRT-PCR and western blotting revealed reduced expression of TPSAB1, IL-4, TNF-α, and CCL2 after ANT treatment. (G) ELISA results further confirmed the suppression of TPS, His, IL-4, TNF-α, and CCL2 secretion in the ANT-treated group. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using 2-tailed Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001. ANT, Antisauvagine; CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor-alpha; TPS, tryptase; TPSAB1, tryptase alpha/beta 1.

Techniques Used: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

SPHK1 mediates the roles of CRFR1 on mast cell activation. (A and B) qRT-PCR and western blotting confirmed the upregulation of SPHK1 in the PDAC mice model. (C) Schematic of the experimental design. PF543, a selective SPHK1 antagonist, or vehicle was administered intraperitoneally to PDAC-bearing mice or mast cell–deficient (C-Kit W-sh ) mice. n = 5 mice per group. (D and E) Von Frey testing and hunching scores demonstrated alleviated cancer-associated abdominal pain after PF543 treatment or mast cell knockout, whereas the analgesic effect of PF543 was abrogated in mast cell deficient mice. (F and G) Inhibition of SPHK1 reduced mRNA and protein expression levels of TPSAB1, IL-4, TNF-α, and CCL2. (H) ELISA showed decreased secretion of mast cell degranulation factors after PF543 treatment, an effect blocked by mast cell knockout. (I and J) SiRNA-1 exhibited the highest knockdown efficiency of SPHK1 at both mRNA and protein levels. (K–M) Knockdown of SPHK1 attenuated CRF-induced expression of mast cell degranulation markers. Data with error bars represented as mean ± SD and analyzed using Student t test (A), 2-way ANOVA (D, E, G, H, K, M), and 1-way ANOVA (I). * P < 0.05, ** P < 0.01, *** P < 0.001. CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; SPHK1, sphingosine kinases type 1; TNF-α, tumor necrosis factor-alpha; TPSAB1, tryptase alpha/beta 1.

Figure Legend Snippet: SPHK1 mediates the roles of CRFR1 on mast cell activation. (A and B) qRT-PCR and western blotting confirmed the upregulation of SPHK1 in the PDAC mice model. (C) Schematic of the experimental design. PF543, a selective SPHK1 antagonist, or vehicle was administered intraperitoneally to PDAC-bearing mice or mast cell–deficient (C-Kit W-sh ) mice. n = 5 mice per group. (D and E) Von Frey testing and hunching scores demonstrated alleviated cancer-associated abdominal pain after PF543 treatment or mast cell knockout, whereas the analgesic effect of PF543 was abrogated in mast cell deficient mice. (F and G) Inhibition of SPHK1 reduced mRNA and protein expression levels of TPSAB1, IL-4, TNF-α, and CCL2. (H) ELISA showed decreased secretion of mast cell degranulation factors after PF543 treatment, an effect blocked by mast cell knockout. (I and J) SiRNA-1 exhibited the highest knockdown efficiency of SPHK1 at both mRNA and protein levels. (K–M) Knockdown of SPHK1 attenuated CRF-induced expression of mast cell degranulation markers. Data with error bars represented as mean ± SD and analyzed using Student t test (A), 2-way ANOVA (D, E, G, H, K, M), and 1-way ANOVA (I). * P < 0.05, ** P < 0.01, *** P < 0.001. CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; SPHK1, sphingosine kinases type 1; TNF-α, tumor necrosis factor-alpha; TPSAB1, tryptase alpha/beta 1.

Techniques Used: Activation Assay, Quantitative RT-PCR, Western Blot, Knock-Out, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Real-time Polymerase Chain Reaction

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Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: Mast cell infiltration and degranulation are enhanced in PDAC with abdominal hyperalgesia. (A) Schematic diagram and representative H&E staining showing the boundary between tumor and para-tumor tissues. Para-tumor regions were defined as areas 4 mm away from the tumor margin. The red dashed line indicated the boundary between tumor and adjacent tissue. Scale bar = 2.5 mm. (B) Representative H&E, toluidine blue, and immunofluorescence staining of tumor and para-tumor tissue sections from paired patients with PDAC with or without abdominal pain (n = 10 vs 10). Black arrows indicate toluidine blue + mast cells. White arrows denote tryptase + CD117 + degranulating mast cells. Scale bar = 50 μm. Green, CD117; Red, tryptase; Blue, DAPI. (C and D) Quantification of toluidine blue + mast cells and tryptase + CD117 + degranulating mast cells demonstrated a significant increase in para-tumor tissues from painful patients with PDAC compared with nonpainful patients. Five random fields of view were selected from para-tumor and tumor regions, analyzed using ImageJ, and averaged. (E) ELISA assays revealed significantly elevated levels of histamine (His), tryptase (TPS), and IL-4 in para-tumor tissues from painful patients with PDAC. (F) ELISA of an additional serum cohort (n = 11 vs 11) verified elevated His, TPS, and IL-4 in painful patients with PDAC. (G) Orthotopic PDAC models were established using Panc-02 cells. The red dashed line indicated the boundary between tumor and para-tumor tissue in mice, with para-tumor area defined as 3 mm away from the tumor margin. (H) The frequency of withdrawal responses to von Frey filament stimulation was significantly increased in orthotopic PDAC models. (I and J) ELISA assays demonstrated elevated levels of His, TPS, and IL-4 in the serum and para-tumor tissues of PDAC models. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (C–F) or Student t test (H–J). * P < 0.05, ** P < 0.01, *** P < 0.001. DAPI, 4',6-Diamidino-2-Phenylindole; ELISA, enzyme-linked immunosorbent assay; His, histamine; IL-4, interleukin-4; NC, normal control mice subjected to sham surgery; PC, orthotopic pancreatic cancer model mice; PDAC, pancreatic ductal adenocarcinoma; TPS, tryptase.

Article Snippet: The murine PDAC cell line Panc-02 and mast cell line MC/9 were obtained from Servicebio Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Control

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: Mast cell CRFR1 expression is elevated in patients with PDAC and mouse models. (A and B) CRF levels increased in both patients with PDAC and mouse models with abdominal hyperalgesia based on ELISA assay. (C) UMAP plot of reanalyzed scRNA-seq data from dataset GSE155698 showing distinct pancreatic cell populations. (D) The percentage of mast cell cluster increased in PDAC tissues compared to normal pancreatic tissues. (E) Average CRFR1 expression per mast cell was significantly elevated in PDAC tissues, whereas CRFR2 expression remained unchanged. (F and G) qRT-PCR and western blotting confirmed the upregulation of CRFR1 in orthotopic PDAC mouse models. (H and I) Flow cytometry demonstrated an increased proportion of CD117 + FcεR + mast cells (gated within CD45 + leukocytes) and elevated CRFR1 expression in CD117 + FcεR + CD45 + mast cells in PDAC mice. (J–M) Immunofluorescence staining further confirmed elevated CRFR1 expression in tryptase + mast cells in PDAC experimental mice and painful human patients with PDAC. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (A, M) or Student t test (B, F, I, K). * P < 0.05, ** P < 0.01, *** P < 0.001. CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; UMAP, uniform manifold approximation and projection.

Article Snippet: The murine PDAC cell line Panc-02 and mast cell line MC/9 were obtained from Servicebio Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Immunofluorescence, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: CRFR1 promotes the activation of mast cell and enhances abdominal pain sensitivity. (A) qRT-PCR verified that CRF (1 μM) significantly upregulated mast cell activation markers, including TPSAB1, IL-4, TNF-α, and CCL2. (B) Western blotting confirmed the increased protein expression of TPSAB1 after CRF stimulation. (C) ELISA assays demonstrated elevated levels of secreted TPS, His, IL-4, TNF-α, and CCL2 in the supernatant of CRF-treated cells. (D) Administration of ANT (a CRFR1 antagonist) significantly reduced abdominal withdrawal response frequency in PDAC mice. (E and F) qRT-PCR and western blotting revealed reduced expression of TPSAB1, IL-4, TNF-α, and CCL2 after ANT treatment. (G) ELISA results further confirmed the suppression of TPS, His, IL-4, TNF-α, and CCL2 secretion in the ANT-treated group. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using 2-tailed Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001. ANT, Antisauvagine; CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor-alpha; TPS, tryptase; TPSAB1, tryptase alpha/beta 1.

Article Snippet: The murine PDAC cell line Panc-02 and mast cell line MC/9 were obtained from Servicebio Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: SPHK1 mediates the roles of CRFR1 on mast cell activation. (A and B) qRT-PCR and western blotting confirmed the upregulation of SPHK1 in the PDAC mice model. (C) Schematic of the experimental design. PF543, a selective SPHK1 antagonist, or vehicle was administered intraperitoneally to PDAC-bearing mice or mast cell–deficient (C-Kit W-sh ) mice. n = 5 mice per group. (D and E) Von Frey testing and hunching scores demonstrated alleviated cancer-associated abdominal pain after PF543 treatment or mast cell knockout, whereas the analgesic effect of PF543 was abrogated in mast cell deficient mice. (F and G) Inhibition of SPHK1 reduced mRNA and protein expression levels of TPSAB1, IL-4, TNF-α, and CCL2. (H) ELISA showed decreased secretion of mast cell degranulation factors after PF543 treatment, an effect blocked by mast cell knockout. (I and J) SiRNA-1 exhibited the highest knockdown efficiency of SPHK1 at both mRNA and protein levels. (K–M) Knockdown of SPHK1 attenuated CRF-induced expression of mast cell degranulation markers. Data with error bars represented as mean ± SD and analyzed using Student t test (A), 2-way ANOVA (D, E, G, H, K, M), and 1-way ANOVA (I). * P < 0.05, ** P < 0.01, *** P < 0.001. CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; SPHK1, sphingosine kinases type 1; TNF-α, tumor necrosis factor-alpha; TPSAB1, tryptase alpha/beta 1.

Article Snippet: The murine PDAC cell line Panc-02 and mast cell line MC/9 were obtained from Servicebio Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Knock-Out, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Real-time Polymerase Chain Reaction

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