Non-invasive Multi-Electrode Array.

<p>The MEA has 16 electrodes placed 2 mm apart and is arranged in 4 rows. Pulses are administered in a sequence that utilizes 4 electrodes at a time, forming 2×2 mm squares (9 total squares). Pulses are applied in pairs, in two directions, perpendicular to each other (18 pulses) for 4 rounds of pulsing (72 total pulses). This image is reprinted from The Journal of Controlled Release doi:10.1016/j.jconrel.2011.01.014 Siqi Guo, Amy Donate, Guarav Basu, Cathryn Lundberg, Loree Heller, Richard Heller “Electro-gene transfer to the skin using a non-invasive multi-electrode array” with permission from Elsevier.</p

Visual assessment of skin damage and healing.

<p>Guinea pigs were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019181#s2" target="_blank">Methods</a> 2.1 with pHBsAg with or without EP. Images were taken of skin pre treatment, immediately post treatment, and at 24, 48, 72, 96 hours and at 7 days. Arrows indicate the treatment sites.</p

KDM2A interacts with Rb and E2F1 in a cell cycle specific manner.

<p>(A) GST pull down assay showing KDM2A binding to Rb and E2F1 <i>in vitro</i>. ³⁵S-lysate lane has 1/10^th protein loaded. (B) MCF-7 cells were serum starved for 48 hr and serum stimulated for 2 hr, 4 hr, 6 hr, 8 hr and 18 hr. Western blotting showing protein expression of KDM2A, Rb, E2F1 and actin. There is no significant change in the expression of KDM2A and E2F1 at different time points when normalized to actin levels. Rb shows increasing hyperphosphorylation from 6 hr to 18 hr of serum stimulation. (C) KDM2A interacts with Rb and E2F1 <i>in vivo</i> in MCF-7 cells as seen by immunoprecipitation-western blot experiment. Interaction of KDM2A with E2F1 decreases at 8 hr of serum stimulation. (D) Chromatin Immunoprecipitation (ChIP) assays showing KDM2A occupancy on E2F1-regulated proliferative promoters, CDC25A and TS. KDM2A occupies the promoters at all time points from 0 hr to 6 hr, reduction is seen at 8 hr and complete absence of KDM2A from the promoters is observed at 18 hr of serum stimulation. (E) KDM2A significantly represses E2F1-mediated E2.Luc transcription in a dose-dependent manner in Renilla luciferase assay.</p

KDM2A represses transcriptional activity of MMPs and VEGF receptors by inhibiting E2F1-mediated transcription.

<p>(A) ChIP assay demonstrates occupancy of KDM2A on MMP9, -14, -15, KDR and FLT-1 similar to E2F1. (B, C, D, E) Transient transfection experiments in MCF-7 cells showed that E2F1 induces MMP2 (B), MMP-9 (C), MMP-14 (D) and MMP-15 (E) promoters, and this was repressed by co-transfection of KDM2A or Rb large pocket. (F, G) Transient transfection experiments in MDA-MB-231 cells demonstrate that KDM2A represses the transcriptional activity of E2F1 on KDR (F) and FLT-1 (G) promoters.</p

Myoepithelial cells of the breast express KDM2A.

<p>(A) Immunohistochemical staining of KDM2A on human breast cancer tissue microarray. Immunostaining was performed using rabbit anti-human KDM2A antibody and representative images of KDM2A expression in normal breast, Ductal Carcinoma in situ, Infiltrating Ductal Carcinoma with and without metastasis, lymph node metastasis are shown. Magnification is 200X, scale bar  = 50 µm. (B) Enlarged images of normal and cancer breast tissue sections. Arrows indicate positively stained myoepithelial cells. Scale bar  = 50 µm. (C) Quantitative analysis of KDM2A in breast tissue microarray. The Immunostaining of KDM2A was quantified by using semi quantitative scoring method based on cellularity and intensity of expression. The means of two independent arrays are shown. All <i>p</i>-values were calculated using a two-sided Student <i>t-</i>test.</p

A model depicting KDM2A function in a mammalian cell.

<p>It can be implied that KDM2A regulates Rb-E2F1 function in the progression of the cell cycle. In a quiescent state, KDM2A represses E2F1 functions on various promoters. Addition of VEGF or serum dissociates KDM2A from these promoters facilitating various cellular processes by transcriptional activation leading to enhanced angiogenesis, invasion and migration of cells.</p

KDM2A co-localizes with E2F1 at 6 hr of serum stimulation.

<p>Quiescent MCF-7 cells were serum starved for 48 hr and serum stimulated for 6 hr and 18 hr. Cells were fixed, permeabilized for 5 min with 0.2% Triton X-100/PBS and immunostained for E2F1 (anti-mouse IgG, green) and KDM2A (anti-rabbit IgG, red). Cells were visualized by confocal microscopy. E2F1 was predominantly localized in the nucleus (upper panels), while KDM2A was more ubiquitously distributed in the cells (middle panels). Co-localization of E2F1 and KDM2A was observed in the nucleus at 6 hr of serum stimulation and disappeared totally by 18 hr (right panels). Images were captured at 630X oil using DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope. Scale bar  = 200 µm. Pearson's correlation for co-localization at 6 hr was 1.0.</p

Silencing KDM2A enhances angiogenic tubulogenesis.

<p>(A) HUVEC cells were transfected with either control siRNA or KDM2A siRNA (75 pmol). 24 hr later, cells were plated on matrigel in complete media (asynchronous) or with or without 100 ng/ml VEGF. Images were captured 18 hr later using Leica inverted microscope and representative images are shown. (B) Tubule sprouting points were estimated using Image Pro software. Ablation of KDM2A showed significant increase (2-fold, p<0.01) in the number of sprouting points. (C) Real-Time showing increased mRNA expression of FLT-1 (2.2±0.11-fold, p<0.01) and KDR (1.7±0.9-fold, p<0.05) receptors upon silencing KDM2A expression. Simultaneous decrease in KDM2A mRNA levels (p<0.01) is seen with 75 pmol of KDM2A siRNA compared to control siRNA.</p

KDM2A suppresses invasion and migration of breast cancer cells.

<p>(A) Western blot analysis showing decreased KDM2A levels in T47D and MCF7 cells with two different siRNAs to KDM2A (Ambion and Santa Cruz) compared to control siRNA. (B) Silencing KDM2A by KDM2A siRNA 1 increased invasion in MCF7 cells by 145±23% (p<0.05) and KDM2A siRNA 2 increased invasion by 118±21% (p<0.05) when compared to control siRNA. (C) In T47D cells, KDM2A siRNA 1 enhanced invasion by 48±28% (p<0.1) and KDM2A siRNA 2 by 64±16% (p<0.05) when compared to control siRNA. (D) Both KDM2A siRNA1 (100 pmol) and KDM2A siRNA2 (100 pmol) transfected MCF7 cells migrated into the wound in the presence of serum at 48 hr when compared to control siRNA transfected cells. Serum starved cells did not migrate into the wound with or without KDM2A suppression. (E) T47D cells additionally show significant migration into the wound after transfection with KDM2A siRNA1 and KDM2A siRNA 2 in the presence of serum at 24 hr when compared to control siRNA. Serum starved cells did not migrate into the wound with or without KDM2A suppression. Magnification-200X.</p

Nicotine promotes the growth of Line1 cells.

<p>(A) Nicotine (1 µM) promotes S-phase entry of serum starved Line1 cells in BrdU incorporation assays. (B) Nicotine (1 mg/kg) significantly increases Line1 tumor growth in Balb/c mice when administered by i.p. injection thrice weekly, p = 0.002, n = 10. (C) Nicotine (25 mg/kg/daily) also significantly increases Line1 tumor growth when administered by transdermal patches p = 0.019, n = 14.</p