Article Figures & Data
Figures
- Figure 1.
Mig-7–specific antibody decreases HEC1A endometrial carcinoma cell chemoinvasion. A, overexpression of amino terminus FLAG-tagged Mig-7 significantly increased Matrigel invasion of HT29 cells compared with vector alone stably transfected cells. Columns, mean; bars, SE. Results are after 24 h of invasion done in quadruplicate. B, Mig-7 affinity–purified antibody, but not IgG rabbit isotype antibody, significantly decreased chemoinvasion of HEC1A cells into Matrigel toward HGF (P < 0.01). Ab, antibody; + HGF, bottom wells contained 20 ng/mL HGF. Results shown are after 72 h of invasion and are representative of three independent experiments done in triplicate. Columns, mean; bars, SE. C, representative flow cytometry histograms of control normal rabbit IgG and Mig-7 antibody–treated HEC1A cells stained with YO-PRO-1 and PI. YO-PRO-1 single-positive cells (bottom oval) are apoptotic, and double-positive cells (top oval) are dead. Mean percentages of three samples are shown. D, representative flow cytometry histograms of control IgG and Mig-7 antibody-treated HEC1A cells stained with PI. Cells in G2-M phase are indicated on histograms. Mean percentages of three samples are shown. Flow cytometry experiments were conducted twice with similar results.
- Figure 2.
Antibody or expression of shRNA specific to Mig-7 decreases activity of MT1-MMP. A, representative immunoblot and densitometry results for α2-macroglobulin capture assay of active MT1-MMP in lysates from equal numbers of HEC1A cells treated with normal rabbit IgG or with Mig-7 antibody. All samples were run on the same gel. Mig-7 antibody treatment significantly decreased MT1-MMP activity by 70% compared with IgG treatment (P = 0.0496). Densitometry data are for the upper, captured MT1-MMP band in each lane normalized to its respective tubulin band. Columns, mean; bars, SE. B, densitometry analysis of the upper, captured MT1-MMP band from α2-macroglobulin capture assay in lysates from RL95 cells stably transfected with shRNA 1-3 (decreased Mig-7 expression) and 3-1 (control, endogenous levels of Mig-7 expression). Results are normalized to β-tubulin. RL95 cells expressing shRNA 1-3 showed a 57% decrease in MT1-MMP activity compared with 3-1 control cells. Columns, mean; bars, SE. C, representative flow cytometry histograms of RL95 shRNA 1-3 and 3-1 stably transfected cells stained with YO-PRO-1 and PI. YO-PRO-1 single-positive cells (bottom oval) were apoptotic, and double-positive cells (top oval) were dead. Mean percentages of three samples are shown. D, representative flow cytometry histograms of RL95 shRNA 1-3 and 3-1 stably transfected cells stained with PI for cell cycle analysis. Cells in G2-M phase are indicated on histograms. Mean percentages of three samples are shown. Flow cytometry experiments were conducted twice with similar results.
- Figure 3.
Expression of shRNA specific to Mig-7 decreases S6 kinase, ERK1/2, and Akt phosphorylation in RL95 cells. A, normalized fluorescence intensity from analysis of phosphorylated ERK1/2, Akt, and S6 kinase in RL95 shRNA 1-3 (knockdown) and 3-1 (control) stably transfected cells. Cells were plated and prepared as given in Materials and Methods. 1-3 shRNA–expressing cells, in which levels of Mig-7 are significantly reduced by >50% (2), showed significant phosphorylation decreases in ERK1/2 by 40% (P = 0.002), in Akt by 10% (P = 0.0449), and in S6 kinase by 30% (P = 0.0155) compared with control RL95 cells that express levels of Mig-7 similar to the RL95 parental cell line. B, normalized fluorescence intensities from analysis of phosphorylated PRAS40 and IGF-IR. All phosphorylation fluorescence intensities were normalized to total respective protein fluorescence intensities. These experiments were done in triplicate.
Figure 4.Mig-7 peptides enhance human monocyte killing of MCF-7 breast carcinoma cells. A, MCF-7 cells expressed Mig-7 mRNA by RT-PCR and protein by immunoblot analyses. Experiments were repeated twice. B, human MC cells stimulated with IL-2 and either MUC-1 peptide (0), pooled control peptides (CTL), or pooled Mig-7 peptides (sequences given in Materials and Methods). MC/effector cell lysis of MCF-7 cells as described in Materials and Methods. Note that Mig-7 peptides significantly (P < 0.001) enhanced MC/effector killing of MCF-7 carcinoma cells. Experiments have been repeated thrice in replicates of six for each treatment group. MCs were isolated from two different individuals. C, MC after stimulations to effector cells ratio to MCF-7 cells and respective percentages of MCF-7 cell–specific lysis.
, no peptide; 
, MUC-1 peptide; 
, MUC-1 plus Mig-7 peptides treatment groups. Columns, mean; bars, SE. Experiments were repeated twice with replicates of six for each treatment group. D, TNF-α production by human isolated MC after indicated peptide stimulation. TNF-α production was measured by ELISA assay as described in Materials and Methods after MC were cultured for 8 d with control peptides or with Mig-7 peptides. Assays were done in replicates of six in three independent experiments with identical results.- Figure 5.
Mig-7 expression is specific to breast carcinoma tissue and cell lines. A, representative Mig-7–specific relative RT-PCR of three breast carcinoma cell lines (T47D, MDA-MB453, DU4475) and commercially available RNA from normal breast tissue of three different human subjects without previous histories of cancer. B to D, representative images of Mig-7 antibody immunohistochemistry on breast tissue array from Cybrdi, Inc., as described in Materials and Methods. Core samples from (B) breast carcinoma. Arrows, positive Mig-7 staining. C, representative normal breast tissue immunohistochemistry with Mig-7 antibody, and (D) Representative image of control normal rabbit IgG instead of Mig-7 as primary antibody immunohistochemistry of breast carcinoma tissue section (serial section to that shown in B). Hematoxylin was used to counterstain. Note a lack of specific staining in D compared with B. Images were taken at ×100 magnification with inserts at ×400 magnification. Scale bars, 100 μm for ×100 images and 20 μm for inserts.
- Figure 6.
Stable knockdown of Mig-7 expression in RL95 cells decreases early primary tumor growth in nude mice. A, representative immunoblot of equal amounts of protein lysates from RL95 parental and Mig-7–specific shRNA stably transfected pooled clones 1-3, 3-1, and 4-2 cell lines. Top, after probing with Mig-7 antibody. Blot was reprobed with tubulin antibody to confirm equal loading and transfer. Statistical analyses of 1-3, 3-1, and parental Mig-7 knockdown and sequences of specific shRNAs were shown previously (2) and 4-2 levels of Mig7 protein were equivalent to those found in 1-3 shRNA knockdown of Mig-7 protein. See Materials and Methods for 4-2 shRNA sequence. B, representative graph showing tumor volumes (mm3) measured 13, 15, 18, and 23 d after injection of Matrigel containing RL95 cell lines stably transfected with shRNA constructs 1-3, 4-2, or 3-1 (control) into nude mice (five animals per cell line, as described in Materials and Methods). Note that cells expressing shRNAs 1-3 or 4-2, which significantly knockdown Mig-7 protein levels, showed significant 60% and 40% to 50% (P < 0.05) decreased tumor volume, respectively, at days 13 and 15 of tumor growth. Points, mean; bars, SE. Anova with Tukey's Multiple Comparison Test was used for statistical analyses. This experiment was done twice with similar results.
Volume 8, Issue 8
