MARCKS Effector Domain contains high homology with other putative nuclear localization sequences.

<p>A schematic of the amino acid sequence of the MARCKS ED with alignment to the nuclear localization sequences of DGK-ζ, BASP1, and AKAP12/SSeCKS/Gravin. Blue circles represent lysines, green circles represent phenylalanines, red circles represent serines, while white circles represent other amino acids using single letter abbreviations. The black lines under the MARCKS effector domain amino acid sequence represent sequence homology to DGK-ζ, BASP1, and AKAP12/SSeCKS/Gravin, respectively.</p

MARCKS mutant expression and localization in U87 glioma cells.

<p><b>A)</b> Diagram depicting the domains of the engineered MARCKS wild-type (WT) and effector domain deleted (ΔED) lentiviral constructs. Starting at the N-terminus there is a myristoylation domain (N-MYR), MH2 domain (N-MH2), an effector domain (WT-ED) with amino acid sequence indicated, and lastly a C-terminus V-5 tag. <b>B)</b> Western blot showing doxycycline inducible MARCKS mutant over-expression in U87 cells. Nuclear and cytoplasmic fractions were prepared and separated by SDS-PAGE and probed for V-5 (for MARCKS expression), lamin and tubulin for nuclear and cytoplasmic fraction, respectively. <b>C)</b> Confocal microscopy is shown for U87 WT-MARCKS (WT) and ΔED-MARCKS (ΔED) cells were fixed and stained for MARCKS (V5), nucleus (DAPI), and PIP₂ with the merged image shown on the right. <b>D)</b> High magnification of the PIP2 staining of the nucleus with punctate staining marked with red arrows.</p

Changes in RNA expression and protein levels with ED-deleted MARCKS.

<p><b>A)</b> After overnight doxycycline-induction of ΔED-MARCKS or WT-MARCKS U87 cells, RNA was collected and ran on NanoString nCounter GX Human Cancer Reference Kit. Data was analyzed on nSolver software. Violin plot displays > 1.5 log fold increase in GBM cells overexpressing an ΔED-MARCKS protein as compared to WT MARCKS. <b>B)</b> Changes in total protein levels as well as phosphorylated protein levels between the ΔED and WT- MARCKS expressing U87 GBM cell lines were determined by Kinex KAM-850 microarray. Data is displayed as z-ratio of ΔED results compared to WT-MARCKS results. Increase in z-ratio is indicated in green dots while decrease is indicated in red dots. Total protein targets are listed in standard font while phosphorylated proteins are listed in italicized font with phosphorylated amino acids designated.</p

MARCKS localizes to the nucleus in D54 and U251 glioma cells.

<p><b>A)</b> D54 and U251 cells were fixed and stained for MARCKS and DAPI with the merged image shown on the right side as indicated. <b>B)</b> D54 and U251 cell lysates were separated into nuclear and cytoplasmic fractions. The proteins were then were separated by 8% SDS-PAGE and probed for MARCKS, the cytoplasmic marker tubulin, and the nuclear marker, lamin.</p

Data_Sheet_1_PARP1 Is Up-Regulated in Non-small Cell Lung Cancer Tissues in the Presence of the Cyanobacterial Toxin Microcystin.pdf

<p>Non-small cell lung cancer (NSCLC) is the major form of lung cancer, with adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being its major subtypes. Smoking alone cannot completely explain the lung cancer etiology. We hypothesize that altered lung microbiome and chronic inflammatory insults in lung tissues contribute to carcinogenesis. Here we explore the microbiome composition of LUAD samples, compared to LUSC and normal samples. Extraction of microbiome DNA in formalin-fixed, paraffin-embedded (FFPE) lung tumor and normal adjacent tissues was meticulously performed. The 16S rRNA product from extracted microbiota was subjected to microbiome amplicon sequencing. To assess the contribution of the host genome, CD36 expression levels were analyzed then integrated with altered NSCLC subtype-specific microbe sequence data. Surprisingly phylum Cyanobacteria was consistently observed in LUAD samples. Across the NSCLC subtypes, differential abundance across four phyla (Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes) was identified based on the univariate analysis (p-value < 6.4e-4 to 3.2e-2). In silico metagenomic and pathway analyses show that presence of microcystin correlates with reduced CD36 and increased PARP1 levels. This was confirmed in microcystin challenged NSCLC (A427) cell lines and Cyanobacteria positive LUAD tissues. Controlling the influx of Cyanobacteria-like particles or microcystin and the inhibition of PARP1 can provide a potential targeted therapy and prevention of inflammation-associated lung carcinogenesis.</p