Basal kinomic activity profiles.

<p>Unsupervised hierarchical clustering of basal (untreated) tyrosine kinomic profiles displaying log transformed slope-exposure for (<b>A</b>) all 144 peptides and (<b>B</b>) as change from sample mean and filtered for variance >1. Red in (A) indicates relative increased signal and in (B) indicates an increase from sample mean. Blue indicates the opposite. Blue arrowhead points to red line denoting dendrogram separation. (<b>C</b>) Western blotting of GAPDH and Actin is shown with sample concentration indicated for each patient.</p

Kinomic platform and Electromagnetic Navigation Bronchoscopy.

<p>(A) Overall experimental flow with a (B) Representative in-procedure display of ENB and a schematic of PamChip assay used to measure basal kinomic activity displayed as (C) raw array picture of the 144 phosphorylatable peptides and (D) phosphorylation changes with drug treatment displayed with illustration of comparative fluorescent detection below.</p

<i>Ex vivo</i> drug response profile.

<p>Displays <i>ex</i><i>vivo</i> drug response profiles as (A) a heatmap of kinase activity (log signal values) change from untreated, clustered by row, of altered phosphopeptides per patient, per dose at 20 nM, 0.5 µM or 20 µM. (B) <i>Ex vivo</i> prewash kinetic peptide phosphorylation (y axis per cell) over time (x axis per cell) of selected peptides in the selected samples, in response to indicated drugs at 20 µM. Blue lines denote untreated, and green lines indicate treated phosphorylation curves.</p

CONSORT diagram on patient enrollment.

<p>CONSORT diagram on patient enrollment.</p

Patient characteristics and tumor evaluation.

<p>M-male; F-female; NSCLC-non small cell lung cancer; MD-PD-moderately differentiated to poorly differentiated; SBRT-stereotactic body radiotherapy.</p><p>Patient characteristics and tumor evaluation.</p

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