MOESM1 of 14-3-3ζ loss leads to neonatal lethality by microRNA-126 downregulation-mediated developmental defects in lung vasculature

Additional file 1: Figure S1. Characterization of the ES cell line RRR334. A RT-PCR to confirm that the cell line traps 14-3-3ζ schematic view of the integration of the gene trap vector in the 14-3-3ζ gene as described in the legend of Fig. 1. The arrowheads indicate the primers for PCR. Endogenous 14-3-3ζ is expressed both in wild-type ES cell control, TC1, and the mutant cell line RRR334. The exogenous mutant allele exists only in the RRR334 cell line. B Determination of the integration site of the gene trap vector using PCR. Arrowheads indicate the primers for PCR. The numbers on each lane of the gel indicate the primer position in the 14-3-3ζ gene. “N” indicates negative control; “M” indicates marker. The 1636 and 506-bp marker sizes are shown. C Western blot analysis of 14-3-3ζ expression level in 8 week old female B6/129 mice mammary gland. Quantification of relative 14-3-3ζ expression level is shown below the 14-3-3ζ blot panel. Figure S2. Characterization of truncated 14-3-3ζ. A Western blot of lysate of MCF7 vector control transfectants (Vc) and two MCF7 transfectants of HA-tagged N-terminal fragment 139 amino acids of 14-3-3ζ [C-terminal deletion (ΔC1 and ΔC12)]. Cells were treated with DMSO or 50 nM MG132 for 4 h. Endogenous 14-3-3ζ was detected using 14-3-3ζ antibody, while the exogenous 14-3-3ζ C-terminal deletion fragment was detected using HA antibody. B 14-3-3ζ N-terminal fragment did not affect p-Mek1 and p-Akt levels. Western blot on lysates from the indicated transfectants were performed with indicated antibodies. β-Actin was used as loading control. C 14-3-3ζ N-terminal fragment did not affect proliferation in MCF-7 cells. MTT assay was performed on the three indicated transfectants. OD was measured at 570 nm and normalized to 650 nm. Figure S3. 14-3-3ζ expression in FVB/NJ and CD-1 14-3-3ζ+/+ and 14-3-3ζ−/− mice. A Analysis of 14-3-3ζ and β-actin in different organs in the CD-1 14-3-3ζ+/+ and 14-3-3ζ−/− mice by western blotting. Quantification of relative 14-3-3ζ expression level is shown below the western panel. B Analysis of 14-3-3ζ and β-actin in different organs in the FVB/NJ 14-3-3ζ+/+ and 14-3-3ζ−/− mice by western blotting. Quantification of relative 14-3-3ζ expression level is shown below the western panel. C Analysis of 14-3-3ζ, 14-3-3β, 14-3-3ε and β-actin in the liver, kidney and lungs from FVB/NJ 14-3-3ζ+/+ and 14-3-3ζ−/− mice by western blotting. Quantification of relative 14-3-3ζ, 14-3-3β, 14-3-3ε expression level is shown below the western panel

Knocking down of p16 enabled the HPNE/K-ras cells to overcome senescence.

<p>(A) Representative fields of senescence-associated β-galactosidase (SA-β-gal) activity in HPNE cell lines. (B) Quantification of SA-β-gal staining in HPNE cells, data are showed as percentage of positive cells **<i>P</i><0.01 versus the corresponding control groups. (C) The expression of cyclin-dependent inhibitors – p15, p21 and p27 was analyzed by Western blot in HPNE cell lines. β-Actin was used as loading control.</p

HPNE cells with activation of K-ras and inactivation of p16 induced tumorigenesis <i>in vivo</i>.

<p>(A) <i>In vivo</i> bioluminescence imaging of tumor growth by HPNE, HPNE/K-ras and HPNE/K-ras/p16shRNA cells at 8 weeks' after injection in NOD/SCID mice is shown. (B) The rates of tumor formation and metastasis in NOD/SCID mice. (C) Representative micrographs showing the histology of the orthotopic tumors formed by HPNE/K-ras/p16shRNA cells as revealed by H&E staining: (i) undifferentiated ductal carcinoma with sarcomatoid features, (ii) necrosis, (iii) liver metastasis, and (iv) spleen metastasis. (D) Immunohistochemical analysis of the expression of HER-2 and EGFR in tumors formed <i>in vivo</i> by the HPNE/K-ras/p16shRNA cells compared with those in human pancreatic cancer and human normal pancreatic duct. Scale bar: 100 µm.</p

Molecular analysis of HPNE cell lines offers insight into malignant transformation in human pancreatic cancer.

<p>(A) Activation of the Akt signaling pathway by K-ras in HPNE cell lines as detected by Western blot analysis. (B) Activation of the MAPK signaling pathway by K-ras in HPNE cell lines. The expression of total and phosphorylated-Erk and p38 was detected by Western blot analysis. β-Actin was used as loading control. (C) Expression of p16 was decreased after siRNA depletion of HBP1, as detected by real-time PCR. (D) Relative quantification of SA-β-gal staining cells after siRNA depletion of HBP1 in HPNE/K-ras cells (E) Expression of EGF and TGFα was increased in HPNE/Kras cells as detected by real-time PCR. (F) Expression of TGFα was increased in HPNE-iKras cells as detected by real-time PCR. (G) The proposed model for transformation of HPNE cell line. hTERT enabled cell to acquire replicating immortality; oncogenic K-ras rendered HPNE cells able to sustain proliferative signaling, and thus increased cell proliferation and cell growth; and inactivation of p16 by shRNA silencing enabled HPNE cells to evade growth suppressors, disrupt the senescence checkpoint, and ultimately induce transformation of HPNE cells.</p

Activation of K-ras and silencing of p16 in HPNE cells increased cell proliferation and growth.

<p>(A) The cell growth curve of HPNE cell lines as detected by using a cell counter. (B) Cell cycle analysis of HPNE cell lines. The percentage of cells in each phase of the cell cycle is shown. (C) Anchorage-independent cell growth of HPNE cell lines in soft agar assay. A representative field of soft agar for each cell line is shown. (D) Cells were incubated with gemcitabine for 72 h, and cell viability was measured by MTT assay. (E) The expression of cell cycle proteins cyclins and c-Myc was increased by mutant K-ras in HPNE cell lines, as determined by Western blot analysis. β-Actin was used as loading control. (F) Relative mRNA levels of E2F target genes in HPNE/K-ras and HPNE/K-ras/p16sh cells. All data are presented as mean ± SD (n = 3 independent experiments). **<i>P</i><0.01 versus the corresponding control groups.</p

Activation of K-ras in HPNE cells increased cell invasion.

<p>(A) The quantification of stained HPNE cell lines per field in a transwell-matrigel penetration assay. Representative micrographs for each cell line are shown. Data are presented as mean ± SD (n = 3 independent experiments). **<i>P</i><0.01 versus the HPNE control cells. (B) The expression of EMT markers cytokeratin-19, E-cadherin, vimentin, and N-cadherin was detected in HPNE cell lines by Western blot analysis. (C) The expression of invasion-related proteins MMP2 and uPA in HPNE cell lines was detected by Western blot analysis. β-Actin was used as loading control.</p

Inhibition of Type I Insulin-Like Growth Factor Receptor Signaling Attenuates the Development of Breast Cancer Brain Metastasis

<div><p>Brain metastasis is a common cause of mortality in cancer patients, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF-IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that IGF-IR is constitutively autophosphorylated in brain-seeking breast cancer sublines. Knockdown of IGF-IR results in a decrease of phospho-AKT and phospho-p70s6k, as well as decreased migration and invasion of MDA-MB-231Br brain-seeking cells. In addition, transient ablation of IGFBP3, which is overexpressed in brain-seeking cells, blocks IGF-IR activation. Using an <i>in vivo</i> experimental brain metastasis model, we show that IGF-IR knockdown brain-seeking cells have reduced potential to establish brain metastases. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.</p> </div

IGF-IR knockdown in brain-seeking breast cancer cells suppresses proliferation, invasion and migration <i>in vitro</i>.

<p>A, Immunoblot of IGF-IRβ and AKT total and phospho-Ser473 expression in 231Br cells stably transfected with control shRNA (vector) or IGF-IRβ shRNA (shIGF-IR B and F clones). B, MTT assay of control and IGF-IR beta knockdown cells at 24, 48 and 72 hr. Values represent mean ± SEM. C, Vector control and shIGF-IR 231Br cells were seeded 100,000 cells per well and were counted after 72 hr. D, Wound-healing assay of vector and shIGF-IR 231Br cells. Images are representative of triplicates at 0 and 21 hr. E, Quantitative measurement of wound closure area from (D). Data were calculated from one representative experiment out of three performed. F, Matrigel invasion assay of vector and shIGF-IR 231Br cells performed in triplicate over 24 hr with complete medium as a chemoattractant. G, Quantitative analysis results of one representative experiment out of three performed in triplicate from (F). Values represent mean ± SEM.</p

IGF-IR knockdown delays brain metastasis and prolongs survival <i>in vivo</i>.

<p>A, Survival curve of mice injected with 231Br cells stably expressing IGF-IR shRNA or vector shRNA. Mice were monitored weekly and sacrificed when moribund. shIGF-IR(B) and shIGF-IR(F) groups had significantly longer survival, p = 0.0012 and p = 0.0133, respectively. B, Median survival of each group from (A). C, H & E and IHC staining of representative brain metastases from each group. H&E panels: dark red = tumor tissue; blue = nucleus; light red = negative. IGF-IR and AKT-pSer473 panels: red = positive; blue = nucleus. GFAP: dark red/brown = positive; blue = nucleus; black arrows = tumor cells; white arrows = tumor-infiltrating astrocytes. Images were taken at 400x magnification.</p

IGFBP3 overexpression contributes to IGF-IR activation in brain seeking cells.

<p>A, Real-time quantitative RT-PCR of IGFBP3 in 231P and 231Br cells. Data are expressed as relative expression as a ratio to housekeeping gene HPRT1 expression. B, Western blot analysis of secreted IGFBP3 protein in the conditioned medium of 231P and 231Br cells. Equal cell numbers were incubated in serum-free medium for 48 hr, and then the conditioned medium was collected and concentrated by 40-fold. C, Western blot analysis of IGFBP3 protein in lysates of 231P and 231Br cells. D, Conditioned medium of 231Br cells transiently transfected with control or IGFBP3 siRNAs for 48 hr. Medium was concentrated by 40-fold and the protein expression of IGFBP3 was analyzed using Western blot. E, IGFBP3 knockdown downregulates IGF-IR phosphorylation. Cells were transfected with either control or IGFBP3 siRNAs. IGF-IR was immunoprecipitated (IP) and immunoblotted with phospho-Tyr antibody. Whole cell lysate (WCL) was used as input control. F, Flow cytometric analysis of 231Br cells after IGFBP3 knockdown. Cells were transfected with either control or IGFBP3 siRNAs, and stained with AlexaFluor 647-phospho Y1131 IGF-IR antibody. IGF-IR phosphorylation decreased in the siRNA groups.</p