Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 is activated in mouse PNETs, and its inhibition improves survival time and suppresses tumor growth in a PNET model . Control pancreas and PNETs were stained for phospho-MK2 ( A ), where was highly expressed as shown by a representative image. The RipTag2 mice were treated as shown in a timeline ( B ) where the sequence of procedures and treatments in the transgenic RipTag2 mice model of PNETs were presented. Furthermore, immunofluorescence staining ( C ) and Kaplan–Meier curve ( D ) show decreased activity of MK2 and increased survival time in MK2 inhibitor-treated mice (MK2i, red), and the weight of PNETs ( E ) were decreased in mice treated with MK2 inhibitor (n = 14 in each group). In PNETs, the gene expression ( F ) of Adgre1 , Casp3 , Fasl and Nos2 are increased while Arg1 is decreased in mice treated with MK2 inhibitor, (n = 6 in each group). PNETs were stained for phospho-MK2 and F4/80 where co-expression of MK2 and macrophage marker was shown by a representative image ( G ). Data are presented as means ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Inhibition, Control, Staining, Sequencing, Transgenic Assay, Immunofluorescence, Activity Assay, Gene Expression, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 inhibition affects production and secretion of cytokines and chemokines in PNETs. Tumor supernatants were examined by multiplex array for the concentration of G-CSF ( A ), IL-6 ( B ), IL-10 ( C ), IL-12 ( D ), KC ( E ), LIF ( F ), MCP-1 ( G ), MIP-1α ( H ), MIP-1β ( I ), MIP-2 ( J) and VEGF ( K ) from tumors obtained from RipTag2 mice treated with DMSO (control, blue) and MK2 inhibitor (MK2i, red). Data are presented as means ± SEM; * p < 0.05 vs. control; n = 6 in each group.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Inhibition, Multiplex Assay, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 inhibition suppress production of metabolic factors in PNETs. Tumor supernatants were examined by multiplex array for the concentration of GCG ( A ), GIP ( B ), GLP-1 ( C ), IAPP ( D ), INS ( E ), LEP ( F ), PP ( G ), PYY ( H ) and RETN ( I ) from tumors obtained from RipTag2 mice treated with DMSO (control, blue) and MK2 inhibitor (MK2i, red). Data are presented as means ± SEM; * p < 0.05 vs. control; n = 6 in each group.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Inhibition, Multiplex Assay, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 KO BMMs are anti-tumorigenic in vitro and in vivo. In vitro studies in the transgenic and the RipTag2 cell-derived allograft tumor models were performed ( A ). Tumor cells ( B ) were plated and cultured from a dissociated RipTag2 tumor. Cell supernatants were examined with a multiplex array for the concentration of CPEP, IAPP and INS ( C ), GCG, GIP, GLP-1, RETN and SCT ( D ). BMMs from WT and MK2 KO mice were cultured, and supernatants examined for the level of IL-10 ( E ) and IL-12 ( F ) by multiplex array after incubation with LPS for 24 h. The percent of annexin V + cells of gated tumor cells was analyzed ( G ) in co-cultures, n = 8 in each group. Representative histograms ( H ) of the flow cytometry analysis of annexin V in the RipTag2 cells treated with activated WT and MK2 KO BMMs. Data are presented as means ± SEM; * p < 0.05 vs. WT BMMs + LPS; *** p < 0.001 vs. RipTag2 cells; ### p < 0.001 vs. RipTag2 cells + non. activated WT or MK2 KO BMMs; $$ p < 0.01 vs. RipTag2 cells + activated WT BMMs.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: In Vitro, In Vivo, Transgenic Assay, Derivative Assay, Cell Culture, Multiplex Assay, Concentration Assay, Incubation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: Adoptive transfer of MK2 KO macrophages improves survival of RipTag2 mice and suppress growth of cell line allograft tumors . The RipTag2 mice were treated with macrophages as shown in a timeline ( A ). The Kaplan–Meier curve ( B ) shows improved survival of mice receiving MK2 KO macrophages compared to WT macrophages, n = 8 in each group. Rag1 KO mice receiving RipTag2 cells were treated with macrophages as presented in the timeline ( C ), and showed decreased tumor growth ( D , E ) with adoptive transfer of MK2 KO macrophages, and these tumors had increased expression of apoptotic genes ( F ) such as Casp3 and Fasl along with decreased expression of Il-10 and increased expression of Il-12 and Nos2 as indicators of macrophage activity, n = 8 in each group. Immunofluorescence staining of NOS2 ( G ) in tumors treated with MK2 KO macrophages shows increased activity of macrophages compared to tumors treated with WT macrophages, n = 11 in each group. Data are presented as means ± SEM; * p < 0.05, *** p < 0.001 vs. control; # p < 0.05, ### p < 0.001 vs. + WT BMMs.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Adoptive Transfer Assay, Expressing, Activity Assay, Immunofluorescence, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 KO macrophages inhibit production and secretion of cytokines/chemokines and metabolic factors in PNETs. Tumor supernatants were examined by multiplex array for the concentration of IL-1β ( A ), IL-6 ( B ), IL-10 ( C ), IL-12 ( D ), MCP-1 ( E ), TNF-α ( F ), INS ( G ), LEP ( H ) and RETN ( I ) in media from the RipTag2 cell-derived allograft tumors obtained from mice treated with PBS (control, black), WT BMMs (+WT BMMs, blue) and MK2 KO BMMs (+MK2 KO BMMs, red). Data are presented as means ± SEM; * p < 0.05 vs. control; # p < 0.05, ## p < 0.01 vs. + WT BMMs; n = 8 in each group.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Multiplex Assay, Concentration Assay, Derivative Assay, Control

Journal: Current Issues in Molecular Biology

Article Title: Mitigation of 3.5 GHz Electromagnetic Field-Induced BV2 Microglial Cytotoxicity by Polydeoxyribonucleotide

doi: 10.3390/cimb47060386

Figure Lengend Snippet: Effect of SP600125, SB203580, or PD98059 on 3.5 GHz EMF-induced growth suppression of BV2 cells and phosphorylation of JNK-1/2, p38 MAPK (MK2), or ERK-1/2 in BV2 cells. ( A ) BV2 cells were exposed to 3.5 GHz EMFs without or with SP600125 (25 μM), a JNK-1/2 inhibitor, SB203580 (25 μM), a p38 MAPK inhibitor, or PD98059 (50 μM), an ERK-1/2 inhibitor for 2 h. The number of surviving cells was analyzed using cell count analysis. Data represent the mean ± SE of three independent experiments. * p < 0.01 compared with the values of the control (no 3.5 GHz EMF exposure). # p < 0.01 compared with the values of 3.5 GHz EMF exposure (no drug), as determined by one-way ANOVA followed by Sidak’s post hoc test. ( B ) A representative image of morphological changes in the conditioned cells in ( A ). ( C ) BV2 cells were exposed to 3.5 GHz EMFs without or with SP600125 (25 μM), SB203580 (25 μM), or PD98059 (50 μM) for 2 h. Whole-cell lysates from the conditioned cells were prepared and analyzed using Western blotting with antibodies.

Article Snippet: The primary antibodies, including eukaryotic initiation factor-2α (eIF-2α) (cat. no. 9722), phosphorylated (p)-extracellular signal-regulated protein kinase-1/2 (p-ERK-1/2) (T202/Y204) (cat. no. 9101), ERK-1/2 (cat. no. 9102), p-JNK-1/2 (T183/Y185) (cat. no. 9251), JNK-1/2 (cat. no. 9252), and p-p38 MAPK (T180/Y182) (cat. no. 9211) p38 MAPK (cat. no. 9212), p-MK2 (T334) (cat. no. 3007), and T-MK2 (cat. no. 3042), were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Phospho-proteomics, Cell Counting, Control, Western Blot

Journal: Science signaling

Article Title: Control of IL-17 receptor signaling and tissue inflammation by the p38α–MKP-1 signaling axis in a mouse model of multiple sclerosis

doi: 10.1126/scisignal.aaa2147

Figure Lengend Snippet: (A) MEFs isolated from WT or p38αCreER mice were treated with 4-OHT, and were left untreated or treated with IL-17 or TNF-α alone or in combination for 24 hours. Cell culture medium was then analyzed to determine the amounts of CXCL1 protein secreted by the cells. Data are means ± SEM of six mice per group. (B) Astrocytes isolated from WT or p38αCreER mice were treated with 4-OHT, and were then incubated in the absence or presence of IL-17 for 5 hours. Cells were subjected to real-time PCR analysis to determine the relative abundances of the indicated mRNAs. Data are means ± SEM of three to seven mice per group. (C) Left: Astrocytes isolated from WT or p38αCreER mice were treated with 4-OHT, and were then incubated in the absence or presence of IL-17 for the indicated times. Samples were then analyzed by Western blotting with antibodies specific for the indicated proteins. Blots are representative of five mice per group from two independent experiments. Right: Relative band intensities were determined by densitometry and are shown as means ± SEM of five mice per group. (D) Astrocytes from WT mice were treated with vehicle or an MK2 inhibitor before being stimulated with IL-17 for 5 hours. Cells were analyzed by real-time PCR to determine the relative abundances of the indicated mRNAs. Data are means ± SEM of seven mice per group. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two to three independent experiments.

Article Snippet: Western blotting analysis was performed as described previously ( 50 ) with antibodies specific for phosphorylated p38 (p-p38) at Thr 180 /Tyr 182 (9211), total p38 (9212), p-MK2 at Thr 334 (27B7), p-CREB at Ser 133 (87G3), p-IκBα (inhibitor of κB α) at Ser 32 (2859), p-ERK at Thr 202 /Tyr 204 (9101), p-JNK at Thr 183 /Tyr 185 (9251, all from Cell Signaling Technology), and β-actin (AC-15, Sigma).

Techniques: Isolation, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Western Blot