Journal: Stem cells (Dayton, Ohio)

Article Title: DAPK1 Interacts with the p38 Isoform MAPK14, Preventing Its Nuclear Translocation and Stimulation of Bone Marrow Adipogenesis.

doi: 10.1093/stmcls/sxac013

Figure Lengend Snippet: Figure 4. The p38 pathway is responsible for DAPK1-related MSC adipogenic differentiation. Intergroup differential volcanic map (A) and the results of GO analysis (B) and KEGG pathway analysis (C) of RNA sequencing data from DAPK1-knockdown and the control MSCs. (D) GSEA identified p38 MAPK pathway from genes of DAPK1-knockdown MSCs and the control MSCs. Normalized enrichment score (NES) and nominal P-value (NOM p-val) are shown. The right panel shows the heat map of gene array in DAPK1-knockdown MSCs (S3) compared with the control (NC) related to p38 MAPK pathway. n = 2 MSCs per group. (E) Pathway inhibitors were used to detect the effects of the enriched pathways by GSEA on DAPK1-related MSC adipogenic differentiation on day 14. Scale bar = 200 μm. (F) Lipid accumulation shown by the ORO staining results in panel E was quantified. (G) The mRNA levels of PPARγ, C/EBPα and FABP4 in DAPK1-knockdown MSCs treated with or without SB203580 in adipogenic differentiation medium for 7 days. (H) Left showed the immunoblot results of activation of major signaling pathways in the DAPK1 knockdown and DAPK1-overexpressing MSCS in adipogenic differentiation medium for 7 days. Right showed the quantification results of immunoblot. (I) Immunoblots for MAPK14, MAPK11, MAPK12, MAPK13, p-p38-T (total p-p38), and GAPDH after IP from MSCs treated with S1 (Si-DAPK1) or the NC as a control with nonspecific IgG (IgG) and anti-p-p38 as indicated. Input: 20 µg of protein of the extracts without IP was loaded. (J) ORO staining and quantification to detect lipid accumulation in DAPK1-knockdown MSCs treated with or without the MAPK14 inhibitor MAPK-IN-1in adipogenic differentiation medium for 14 days. Human MSCs were used in the above tests. Scale bar = 200 μm. Data are presented as the means ± SEMs. *P < .05, **P < .01, ***P < .001. n = 3 independent experiments with 3 different MSCs per group.

Article Snippet: Erlotinib (EGFR pathway inhibitor, HY-50896, MCE), capivasertib (FOXO pathway inhibitor, HY-15431, MCE), SB203580 (p38 MAPK pathway inhibitor, HY-10256, MCE), MAPK-IN-1 (MAPK14 inhibitor, HY-18874, MCE), and MAPK13-IN-1 (MAPK13 inhibitor, HY-18850, MCE) were used in our study.

Techniques: RNA Sequencing, Knockdown, Control, Staining, Western Blot, Activation Assay, Protein-Protein interactions

Journal: Stem cells (Dayton, Ohio)

Article Title: DAPK1 Interacts with the p38 Isoform MAPK14, Preventing Its Nuclear Translocation and Stimulation of Bone Marrow Adipogenesis.

doi: 10.1093/stmcls/sxac013

Figure Lengend Snippet: Figure 5. DAPK1 interacts with MAPK14 in vitro and in vivo. (A) Immunoprecipitates obtained from human MSC extracts (5 µg of protein) with nonspecific IgG or antibody against DAPK1 were stained with Coomassie Blue. (B) The peptide sequences of the DAPK1 and MAPK14 proteins were detected in the co-IP complex. (C) Co-IP of 500 µg of protein from human MSCs with nonspecific IgG (IgG), anti-DAPK1, or anti-MAPK14 as indicated. Input: Twenty micrograms of protein extract without IP was loaded. (D) Colocalization of DAPK1 and MAPK14 in human MSCs. Scale bar = 50 μm. 293T cells transfected with Flag-tagged DAPK1 and HA-tagged MAPK14 were subjected to immunoprecipitation and immunoblot analysis (E) and immunofluorescence doubling staining (F) with the indicated antibodies. Scale bar = 20 μm.

Article Snippet: Erlotinib (EGFR pathway inhibitor, HY-50896, MCE), capivasertib (FOXO pathway inhibitor, HY-15431, MCE), SB203580 (p38 MAPK pathway inhibitor, HY-10256, MCE), MAPK-IN-1 (MAPK14 inhibitor, HY-18874, MCE), and MAPK13-IN-1 (MAPK13 inhibitor, HY-18850, MCE) were used in our study.

Techniques: In Vitro, In Vivo, Staining, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Immunofluorescence