LDH-A is essential for network formation on Matrigel <i>in vitro</i>.

<p>3x10⁴ cells from Tet-On and Tet-Off systems were seeded on Matrigel in 48 wells plate and kept at 37°C overnight. Cells expressing high LDH-A levels from both systems started to form networks as early as two hours after seeding, and complete networks were observed after 24 hours. Cells with low LDH-A levels failed to form networks.</p

LDH-A is required for PMVECs to sustain aerobic glycolysis and rapid growth.

<p>Western blot analysis demonstrates that LDH-A protein decreases in the absence of doxycycline over a 7-day time course (A) resulting in decreased lactate production (B), glucose consumption (C) and PMVEC growth (D). Panel A shows a representative western blot taken from three different experiments performed in parallel with the growth curve. Two-way ANOVA was used to compare between groups, and Bonferroni post hoc test was performed as needed. Significant differences over time (^) and between doxycycline treatments (*) are shown (P < 0.05). Data represent averages <u>+</u> SEM from 3-5 experiments per group, each performed in triplicate.</p

Cells injected in Matrigel are responsible for formation of new blood vessels.

<p>(A) Representative micrographs of GFP-positive endothelial cells. The presence of GFP was detected using anti-GFP antibody followed by biotinylated horse anti-rabbit secondary antibody, visualized using the DAB substrate system. Arrows highlight positive endothelial cell staining in intact vessels. (B) Micrographs depicting negative staining in rat sections using an isotype control antibody. Arrows indicate endothelial cells that do not display staining using the control antibody. All micrographs were taken using a Nikon 80i upright microscope fitted with a 60X objective.</p

Map of the Tet-Off construct and induction of LDH-A shRNA.

<p>(A) PMVECs were infected with a retrovirus, enabling reverse tetracycline-controlled transactivator protein (tTA) expression. Cells were selected to homogeneity using blasticidin, reinfected with a lentivirus [shown in (B)], and selected to homogeneity using puromycin. The resulting double-transfection enabled the expression of a LDH-A short hairpin RNA (shRNA) in the absence of doxycycline. Bsr, blasticidin resistance gene; EGFP, enhanced green fluorescent protein; HIV RRE, human immunodeficiency virus Rev response element; IRES EMV, encephalomyocarditis virus internal ribosome entry site; LTR, retro/lentiviral long terminal repeat; PAC, puromycin resistance gene; PSV40, simian virus 40 promoter; PTet, doxycycline-regulated promoter; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element; mir, 5’–3’ flanking sequence derived from the murine mir (micro-RNA gene)-15. (C) Doxycycline retrieval promoted expression of the red fluorescent protein mCherry in infected PMVECs.</p

Kaplan–Meier incident curve including metastasis, recurrence, and death (all but disease free)

<p><b>Copyright information:</b></p><p>Taken from "Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator"</p><p>Breast Cancer Research 2004;6(5):R478-R487.</p><p>Published online 28 Jun 2004</p><p>PMCID:PMC549164.</p><p>Copyright © 2004 King et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Patients were divided into two groups based on the level of transcripts of activated leukocyte cell adhesion molecule (ALCAM). Pink line, patients with high levels of ALCAM; green line, patients with low levels of ALCAM. Pink line, mean time to incident was 100.5 months. Green line, mean time to incident was 79.6 months. Cox proportion analysis between the two groups, = 0.009

ExoY activity affects microtubule assembly in PMVECs.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺. Untreated control cells (Ctr) and infected cells were then placed on ice to induce microtubule disassembly. Microtubule re-growth was initiated by transferring the cells to 37°C. [<b>A.</b>] Individual coverslips were fixed either at the time of transfer (T = 0) or at varying times after transfer to 37°C. The coverslips then were labeled with antitubulin antibodies. Cells at 4 and 8 minutes post-transfer are shown; microtubule growth is apparent by 4 minutes and peripheral microtubules are resolved by 8 minutes in untreated and K81M infected cells. Microtubule re-growth lagged significantly in cells intoxicated with wt ExoY. Bar = 10 µm. [<b>B.</b>] Polymerized (P) and soluble unpolymerized (S) tubulin levels were quantified by immunoblot analysis using antitubulin antibodies. To obtain soluble and polymer fractions, control cells and cells infected with bacteria expressing either ExoY^K81M or ExoY⁺ were extracted at 8 minutes post-transfer to 37°C. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY [0.10±0.06 <i>vs.</i> 0.36±0.10 (K81M) and 0.41±0.09 (Ctr); n = 4; P<0.05 compared to both K81M and untreated cells]. [<b>C.</b>] Tau levels are unchanged following cold treatment to disassemble microtubules. Cells were treated with cold and then whole extracts were collected from control cells (Ctr) and from cells that were intoxicated with either ExoY⁺ or ExoY^K81M. The extracts were then probed for tau levels using polyclonal anti-tau antibody.</p

ExoY activity does not noticeably affect microtubule disassembly.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M (upper panels) or wild type ExoY (middle panels). Following infection, cells were either fixed before microtubule disassembly was induced (left) or at 1 (middle) or 2 minutes (right) after being placed at 0°C. As shown, disassembly was complete in both control (Ctr), ExoY^K81M and ExoY⁺ expressing cells by 2 minutes after transfer to 0°C. Bar = 10 µm.</p

Phosphorylation causes Tau to dissociate from microtubules.

<p>[<b>A.</b>] Untreated cells (Ctr) as well as cells that were infected with P. aeruginosa expressing either ExoY^K81M or ExoY⁺ were incubated, and then extracts were prepared and soluble fractions and cell ghosts containing intact microtubules were collected. Both microtubule-associated (Mt) and soluble free Tau (S) were assayed by immunoblot using a pan-Tau antibody. Significantly less tau was present in cells ghosts prepared from cells intoxicated with ExoY⁺ compared to those prepared from either untreated or K81M-intoxicated cells [0.22±0.09 <i>vs.</i> 0.44±0.08 (Ctr) and 0.46±0.04 (K81M); n = 4; P<0.05 compared to both K81M and untreated cells]. [<b>B.</b>] <b>Tau co-pellets with taxol-stabilized microtubules.</b> Extracts were prepared from untreated control PMVECs (Ctr) and from cells infected with bacteria expressing either ExoY⁺ (middle) or ExoY^K81M (right), microtubules were assembled by addition of taxol, and then the assembled microtubules were pelleted through a sucrose cushion. The pelleted microtubules were then probed by immunoblot using polyclonal anti-tau antibody (top). The blot was then stripped and re-probed using antitubulin antibody (bottom).</p

Phosphorylation of tau is essential for disrupted microtubule assembly.

<p>Over-expression of a non-phosphorylatable form of Tau (S214A mutant) rescues PMVECs from effects of ExoY⁺ on microtubule assembly. PMVECs were stably transfected with a cDNA encoding a form of Tau mutated at the PKA phosphorylation site and then infected with <i>P. aeruginosa</i> encoding either ExoY^K81M or ExoY⁺. Microtubules were disassembled by incubation on ice, and then microtubule re-assembly was initiated by transferring cells to 37°C. [<b>A.</b>] S214A Tau-expressing control cells (Ctr) and cells infected with either ExoY^K81M or ExoY⁺ were fixed and labeled with antitubulin antibodies either at the time of transfer to 37°C (top) or at 8 minutes post-transfer (bottom). Bar = 10 µm. [<b>B.</b>] Polymerized (P) and soluble unpolymerized (S) tubulin levels were quantified in S214A-expressing cells at 8 minutes post-transfer to 37°C. Extracts were prepared from control cells (Ctr) and from cells infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺. There was no significant difference in the ratio of microtubule polymer to soluble tubulin when cells infected with bacteria expressing ExoY⁺ were compared to those expressing ExoY^K81M or uninfected control cells [0.35±0.8 <i>vs.</i> 0.38±0.06 (K81M) and 0.44±0.11 (Ctr); n = 5].</p

ExoY activity causes a decrease in microtubules in PMVECs.

<p>[<b>A</b>] PMVECs infected with <i>P.aeruginosa</i> expressing either non-functional K81M mutant ExoY (ExoY^K81M; center) or wild type ExoY (right) were observed following processing for anti-tubulin immunofluorescence microscopy. Uninfected cells are also shown (left). Bar = 10 µm. [<b>B</b>] Levels of polymerized tubulin (P) and unpolymerized soluble tubulin (S) were quantified by immunoblot analysis using antibody against α-tubulin. Extracts obtained from untreated cells (Ctr) and from PMVECs infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺ are shown. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY⁺ [0.29±0.06 <i>vs.</i> 0.44±0.05 (ExoY^K81M) and 0.49±0.08 (Ctr); n = 5; P<0.05 compared to both ExoY^K81M and untreated control].</p