Journal: Journal of Leukocyte Biology

Article Title: Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

doi: 10.1189/jlb.1211604

Figure Lengend Snippet: The MANS, but not the RNS, peptide inhibits cell migration. Cells were untreated or treated with 50 μM peptide prior to adding MCP-1 in a Transwell migration assay. Cells that migrated through the pores to the lower side of the insert membrane, were fixed and counted at 40× magnification. That number is given on each panel. Contrast was enhanced for this figure after the panels were assembled to clearly define the migrated cells from the visible membrane pores.

Article Snippet: MANS and RNS peptides were synthesized by Genemed Synthesis (San Antonio, TX, USA).

Techniques: Migration, Transwell Migration Assay, Membrane

Journal: Journal of Leukocyte Biology

Article Title: Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

doi: 10.1189/jlb.1211604

Figure Lengend Snippet: J774A.1 cells were pretreated with 50 μM MANS or RNS. Migration was measured in response to: (A) MCP-1 (50 ng/ml); (B) MCP-1 (100 ng/ml); or (C) C5a (10 ng/ml). Cells were exposed for 3 h, at which time, migrating cells were quantified. Data represent mean ± sem; n = 3; **significantly different from media control; †significantly different from stimulated/media control; P < 0.05.

Article Snippet: MANS and RNS peptides were synthesized by Genemed Synthesis (San Antonio, TX, USA).

Techniques: Migration, Control

Journal: Journal of Leukocyte Biology

Article Title: Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

doi: 10.1189/jlb.1211604

Figure Lengend Snippet: Isolated cells were pretreated with: (A) 25 μM or (B) 50 μM MANS or RNS peptide for 15 min. Migration was measured in response to MCP-1 (100 ng/ml). Cells were exposed for 3 h, at which time, migrating cells were quantified. Data represent mean ± sem; n = 3; **significantly different from media control; †significantly different from MCP-1/media control; P < 0.05.

Article Snippet: MANS and RNS peptides were synthesized by Genemed Synthesis (San Antonio, TX, USA).

Techniques: Isolation, Migration, Control

Journal: Cancers

Article Title: WNT5A-Induced Activation of the Protein Kinase C Substrate MARCKS Is Required for Melanoma Cell Invasion

doi: 10.3390/cancers12020346

Figure Lengend Snippet: MARCKS phosphorylation-specific peptide MANS blocks WNT5A-induced melanoma cell invasion. Western blotting and transwell invasion assays were performed to evaluate the specific role of MARCKS phosphorylation in WNT5A-promoted melanoma cell invasion. ( A ) Western blots showing the expression of total MARCKS and levels of pMARCKS Ser-159/163 in 100 µM RNS- or MANS peptide-treated A2058 melanoma cells. Representative blots of five separate experiments are shown here. ( B,C ) The graph represents the densitometry analysis of ( B ) MARCKS and ( C ) pMARCKS Ser-159/163 levels normalized against total MARCKS levels. The results (n = 5) are presented as the means ± S.E.M.; ***, p < 0.001. ( D ) Effect of the direct inhibition of pMARCKS on WNT5A-induced melanoma cell invasion was evaluated by transwell cell invasion assay as mentioned in the materials and methods section. ( D ) Graph showing the relative melanoma cell invasion of rWNT5A unstimulated/stimulated and RNS peptide- or MANS peptide-treated A2058 melanoma cells. The results (n = 4) are presented as the means ± S.E.M.; *, p < 0.05, and ***, p < 0.001.

Article Snippet: The MANS and RNS peptides were custom synthesized by CASLO ApS (Lyngby, Denmark).

Techniques: Phospho-proteomics, Western Blot, Expressing, Inhibition, Invasion Assay

Journal: Oncotarget

Article Title: Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

doi: 10.18632/oncotarget.11340

Figure Lengend Snippet: ( A ) MARCKS protein expression of CEM and THP-1 cells and their BTZ resistant sublines CEM/BTZ200 and THP-1/BTZ200. In addition, the CEM/BTZ200 cultured in absence of BTZ for several months (CEM/BTZ-200) is also included. Western blot analysis of MARCKS (green) and β-actin (red) as a loading control, (B) Ratio of the quantification of MARCKS protein expression (β-actin normalized) of proteasome inhibitor (PR924 and Marizomib) resistant THP-1 and CEM cells relative to their parental cells. (C) Basal levels of phosphorylated MARCKS expression in CEM/WT and CEM/BTZ cells before and after incubation with PMA (50 nM for 1 hour,), and a combination of PMA and RNS (1 hour 100 nM) or MANS (1 hour 100 μM).

Article Snippet: MARCKS inhibitory peptide (MANS) was from Genemed Synthesis Inc (San Antonio, Texas, USA).

Techniques: Expressing, Cell Culture, Western Blot, Control, Incubation

Journal: Oncotarget

Article Title: Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

doi: 10.18632/oncotarget.11340

Figure Lengend Snippet: ( A ) MARCKS and ubiquitin protein expression in CEM/WT cells, CEM/BTZ7 cells treated with 30 nM BTZ for 24 hours and CEM/BTZ200 cells treated with 400 nM BTZ for 24 hours. Left panel: DAPI nuclear staining (Blue), ubiquitin (Green), and MARCKS (Red), right panel: level of co-localization of MARCKS with ubiquitin as calculated by the SlideBook microscope software as depicted in blue (normal) to magenta (strong co-localization). Single channel images are shown in . ( B ) Ubiquitin (green) and DAPI (blue) staining in HeLa cells after 24 hour incubation with the supernatants of cultures of CEM/WT, CEM/BTZ7 and CEM/BTZ200 cells untreated or treated with the indicated concentrations of BTZ for 1 hour. ( C ) PKH labeling and ubiquitin protein expression in CEM/WT cells, CEM/BTZ7 and CEM/BTZ200 cells treated with the abovementioned concentrations of BTZ for 24 hours. DAPI nuclear staining (Blue), PKH (Green), and ubiquitin (Red) and level of colocalization of MARCKS with ubiquitin as calculated by the SlideBook microscope software as depicted in blue to magenta (Right panel). ( D ) Ubiquitin (red) and PKH (green) staining in HeLa cells after 24 hour incubation with the supernatants of cultures of CEM/WT, CEM/BTZ7 and CEM/BTZ200 cells untreated or treated with the indicated concentrations of BTZ. Calculated co-localization is depicted in yellow. ( E ) Ubiquitin (green) and MARCKS (red) staining in a primary ALL patient samples, one sensitive to BTZ (LC50: BTZ 6 nM) and one resistant to BTZ (LC50 BTZ: 262 nM) ( F ) without and after incubation with low concentration (7 nM) or high concentration of BTZ (400 nM). In the merge figure, the co-localization of the two proteins is shown in yellow. The right top of the figures depict a quantification using a line scan through the corresponding area of interest (α and β).

Article Snippet: MARCKS inhibitory peptide (MANS) was from Genemed Synthesis Inc (San Antonio, Texas, USA).

Techniques: Ubiquitin Proteomics, Expressing, Staining, Microscopy, Software, Incubation, Labeling, Concentration Assay

Journal: Oncotarget

Article Title: Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

doi: 10.18632/oncotarget.11340

Figure Lengend Snippet: CEM/BTZ cells harboring PSMB5 mutations (indicated by *) have a diminished capacity of inhibition of proteasomal catalytic activity by BTZ [ , ]. Upon exposure of CEM/BTZ7 and CEM/BTZ200 cells to BTZ concentrations that block PSMB5 activity (30 nM and 400 nM BTZ, respectively), these cells accumulate polyubiquitinated proteins. This coincides with the biogenesis of vesicle-like structures incorporating these ubiquitinated proteins. These vesicles then traffic along actin/cytoskeleton axis to the plasma membrane with MARCKS protein serving as partner protein. Here myristoylated-anchored MARCKS facilitates exocytosis of vesicles which subsequently can be taken up by recipient (HeLa) cells. Proficient proteasomal activity in recipient cells would allow degradation of ubiquitinated proteins from BTZ-resistant cells. Through a mechanism of exocytosis-mediated extrusion of vesicles containing ubiquitinated proteins, BTZ-resistant cells can overcome proteolytic stress over a broad range of BTZ concentrations.

Article Snippet: MARCKS inhibitory peptide (MANS) was from Genemed Synthesis Inc (San Antonio, Texas, USA).

Techniques: Inhibition, Activity Assay, Blocking Assay, Clinical Proteomics, Membrane

Journal: American Journal of Cancer Research

Article Title: SCF/c-KIT signaling promotes mucus secretion of colonic goblet cells and development of mucinous colorectal adenocarcinoma

doi:

Figure Lengend Snippet: MARCKS accelerated mucus secretion in mouse colon. Alcian blue and PAS staining showing goblet cells and inner mucus layer (arrow) in distal colon of WT mice treated with normal saline, MANS peptide or RNS peptide and additional pilocarpine. WT mice received no any treatment were set as controls. Pilocarpine stimulated mucus secretion a lot in normal saline and RNS mice, indicated by the thick inner mucus layer and nearly empty cytoplasm of goblet cells. However, the effect of pilocarpine was deprived by MANS, indicated by the thin inner mucus layer and filled mucin residual in goblet cells.

Article Snippet: MANS peptide (identical to the first 24 amino acids of the N-terminus of MARCKS; myristic acid-GAQFSKTAAKGEAAAERPGEAAVA) or missense control RNS peptide (myristic acid-GTAPAAEGAGAEVKRASAEAKQAF) (Sangon Biotech, Shanghai, China) were administered at a final concentration of 80 μM (8 mg/kg).

Techniques: Staining, Saline