Additional file 2: Figure S1B. of 3D culture of Her2+ breast cancer cells promotes AKT to MAPK switching and a loss of therapeutic response

Process of recovery of cells from 3D culture for experimental analysis. Cells were recovered from the 3D cultures for counting, immunocytochemical analysis or Western blotting as shown using a modification of a previously reported method (Arnold 2001). (DOC 52 kb

Additional file 3: Figure S2. of 3D culture of Her2+ breast cancer cells promotes AKT to MAPK switching and a loss of therapeutic response

Effects of AKT and MAPK inhibition on apoptosis in BT474 and MDAMB361 cells. Cell lines grown in 2D or 3D culture were exposed to AKT inhibitor (MK-2206) or MEK inhibitor (U0126) prior to cell lysis and Western blotting using an antibody that recognizes full length (116kDa) and cleaved (85kDa) forms of PARP, the latter form apparent upon apoptosis. Neither AKT or MAPK inhibition resulted in a significant loss of full length PARP or a corresponding gain in cleaved PARP for either cell line. In contrast, cleaved PARP was seen to increase in response to the apoptosis inducer, camptothecin, included as a positive control. (DOC 59 kb

FACS analysis of the effects of <i>Zip4</i> knockdown on Hepa cell apoptosis and reentry into the cell cycle after release from hydroxyurea into zinc-deficient medium.

<p>Hepa cells expressing <i>Zip4</i> RNAi (<b>Z4 RNAi</b>) or control RNAi (<b>Con RNAi</b>) from lentivirus vectors were purified for GFP expression by FACS. Confluent cultures were replated at low density (1∶10) in zinc-deficient medium (DMEM plus 10% Chelex-treated FBS), and blocked overnight in G₀/G₁ using hydroxyurea. The medium was replaced with fresh zinc-deficient medium and progression through the cell cycle was quantified at the indicated times after release. Cell cycle parameters were monitored by propidium iodide staining followed by FACS. The percentage of cells in each phase of the cell cycle is presented within each FACS scan. <b>Part A:</b> Cells in G0/G1. <b>Part B:</b> Cells in S phase. <b>Part C:</b> Cells in G2/M. <b>Part D:</b> Cells in the sub-G₁ fraction which is indicative of apoptotic cells. Data are expressed as the mean ± S.D. *Indicates a significant difference between <i>Zip4</i> RNAi and control RNAi (P<0.001).</p

Detection of <i>Zip4</i> mRNA and ZIP4 protein in HCC from <i>FXR</i>-knockout and wild-type mice.

<p><b>Part A:</b> HCC from <i>FXR</i>-knockout mice (<b>FXR KO</b>; 15 months of age) and normal liver from wild-type (<b>WT</b>) litter mates were analyzed by Northern blot hybridization to detect <i>Zip4</i> mRNA in total RNA. <i>Actin</i> mRNA served as a loading control in the Northern blots and staining of 28S and 18S rRNA served as a control for integrity and quantity of RNA loaded. <b>Part B:</b> Immunohistochemical detection of ZIP4 in paraffin sections of HCC from <i>FXR</i>-knockout and wild-type mice. <b>Arrow</b> is pointing to the boundary of a well-circumscribed lesion containing ZIP4-positive hepatocytes in the <i>FXR</i>-knockout liver. Brown deposits indicate ZIP4 staining. Blue color indicates hematoxylin stained nuclei.</p

Increased Expression of <i>Zip4</i> (<i>Slc39a4</i>) May also Occur in Many Different Cancers.

<p>*<i>Zip4</i> mRNA elevated in microarray compared with control. <a href="http://www.ncbi.nlm.nih.gov/geo" target="_blank">http://www.ncbi.nlm.nih.gov/geo</a>.</p><p>**Top ten percent of genes expressed in a truncated listing of tumors as described/listed.</p

Immunofluorescence detection of ZIP4 in human HCC.

<p><b>Part A:</b> Frozen sections of HCC from patients 3, 4 and 11 and normal liver tissue from patient 3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone-0013158-t001" target="_blank">Table 1</a>) were fixed, permeablized and blocked then ZIP4 was detected using an antipeptide antibody against mouse ZIP4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-DufnerBeattie2" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-DufnerBeattie3" target="_blank">[47]</a>. The peptide immunogen is well conserved between mouse and human and specificity of this antibody has been established <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-DufnerBeattie2" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-Weaver1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-Huang1" target="_blank">[46]</a>. Sections were washed and incubated sequentially with biotinylated secondary antibody then QDot 655 streptavidin conjugate. Red indicates the presence of ZIP4 whereas blue indicates nuclei stained with DAPI. Adobe Photoshop image capture software was used to capture these images. <b>Part B:</b> Frozen serial sections from patients 3 & 4 stained with hematoxylin and eosin to reveal the cellular structure of the ZIP4 positive tumors. <b>Arrows</b> point to the boundary of well-circumscribed lesions containing ZIP4 positive hepatocytes.</p

FACS Analysis of the Effects of ZIP4 Knockdown on Hepa cell Apoptosis and Progression after Release from Hydroxyurea Block into Zinc-Deficient Medium^*.

<p>*Details of this experiment are presented in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone-0013158-g004" target="_blank">Figure 4</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#s4" target="_blank">Materials and Methods</a>.</p

Aberrant Expression of <i>Zip4</i> (<i>Slc39a4</i>) in Human HCC.

<p>*Relative <i>Zip4</i> mRNA levels were normalized to that of <i>GAPDH</i> and are presented as 2∧(Ct<i>GAPDH</i>-Ct<i>ZIP4</i>).</p

Effects of RNAi knockdown of <i>Zip4</i> and/or forced expression of <i>Zip4</i> on the migration of Hepa and MCF-7 cells.

<p>Hepa cells expressing <i>Zip4</i> RNAi (<b>Z4 RNAi</b>) or control RNAi (<b>Con RNAi</b>) from lentivirus vectors were purified for GFP expression by FACS. Cells were seeded onto fibronectin-coated Transwell inserts and allowed to migrate for a period of 16 h. Non-migratory cells were removed and cells that had migrated through the membrane were fixed and stained with crystal violet. Cell migration was visualized by microscopy (<b>Part A</b>) and quantified by measuring the absorbance of cell lysates at 595 nm (<b>Part B</b>). In parallel, Hepa cells expressing <i>Zip4</i> RNAi or control RNAi were transfected with a human <i>Zip4</i>-HA expression vector (<b>Z4HA</b>), serum starved overnight and then seeded onto Transwell inserts and allowed to migrate for 16 h. Migratory cells were visualized and quantified as described above. ** Indicates a significant difference from control RNAi and/or <i>Z4</i> RNAi, left two bars (P<0.001) and # indicates a significant difference from control RNAi and/or <i>Z4</i> RNAi/<i>Z4</i>HA (P<0.001). <b>Part C:</b> MCF-7 cells were transfected with an expression vector for mouse <i>Zip4</i>-HA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-Wang1" target="_blank">[33]</a> (<b>Z4HA</b>), or exposed to transfection reagent without vector (<b>control</b>), and then seeded onto fibronectin-coated Transwell inserts. Cells were allowed to migrate for a period of 16 h, non-migratory cells were removed and cells that had migrated through the membrane were fixed and stained. Cell migration was quantified by counting the numbers of cells in 5 randomly-chosen fields of view per membrane (n≥3 for each treatment) and results are plotted as the mean cell migration (percent of control ± S.D.). ** Indicates a significant difference between control and <i>Z4</i>HA transfected MCF-7 cells (P<0.001).</p

Western blot analysis of RNAi knockdown of <i>Zip4</i> in mouse Hepa cells.

<p>RNAi (two RNAi sequences) against mouse <i>Zip4</i> (<b>Z4</b>) and a scrambled RNAi control (<b>Con</b>) were expressed from the U6 promoter in lentivirus vectors that also express GFP driven by the <i>PGK</i> promoter. Cells were infected and then purified for GFP expression by FACS. Lentivirus infected cells and uninfected Hepa cells (<b>-</b>) were then cultured for 24 hr in zinc-adequate medium (<b>N</b>) which contains normal FBS or zinc-deficient medium (<b>CX</b>) which contains Chelex-treated FBS. Membrane preparations from these cells were then fractionated by SDS-PAGE, transferred to a membrane, and ZIP4 was detected using an antipeptide antibody against mouse ZIP4. A prominent ∼73 kDa ZIP4 band was detected (predicted size) as well as a larger ZIP4 aggregate. ZIP1 was detected as a loading control (bottom panel). In Hepa cells a non-specific (<b>NS</b>) band, which is predominately cytosolic, was detected in these membrane preparations.</p