Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.

doi: 10.4049/jimmunol.1202451

Figure Lengend Snippet: FIGURE 1. Characterization of the recom- binant CR1 CCP 22–30_His fragment. (A) SDS-PAGE analysis of 4 mg CR1 CCP22-30 under reducing (R) and nonreducing (NR) conditions. The positions of the m.w. mark- ers are indicated. (B) CD spectroscopy of CR1 CCP22–30. A spectrum was recorded in the far-UV (200–260 nm) and collected six times. The mean values for each wavelength were calculated. The maximal ellipticity is indicated by an arrowhead. (C) Electron microscopy analysis of CR1 CCP22–30 (375000) after negative staining with 2% sodium silicotungstate.

Article Snippet: Recombinant soluble human CR1 was purchased from R&D Systems Europe (Lille, France).

Techniques: SDS Page, Circular Dichroism, Electron Microscopy, Negative Staining

Journal: Oncogene

Article Title: CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

doi: 10.1038/onc.2017.87

Figure Lengend Snippet: Figure 2. Coculture with human osteoblasts (OBs) increases expression of CRIPTO and GRP78 and stem cell-like properties of human PCa cells. (a–c) CRIPTO and GRP78 expression in PC-3M-Pro4Luc2 and C4-2B human PCa cell lines. Error bars ± s.e.m. (d) CRIPTO and GRP78 expression in highly metastatic sub-population of ALDHhigh PCa cells vs low metastatic ALDHlow in PC-3M-Pro4Luc2 cells. Error bars ± s.e.m. (e and f) CRIPTO but not GRP78 protein is increased in highly metastatic sub-population of ALDHhigh PCa cells vs low metastatic ALDHlow in PC-3M-Pro4Luc2 cells isolated after viable cell sorting. (g) Direct coculture of fluorescently labeled PC-3M-Pro4Luc2 human PCa cells (T; 900 000 cells seeded per petri dish) with differentiated human OBs (confluent layer of differentiated cells per petri dish) for 48 h increases the size of the ALDHhigh sub- population in PCa cells. Each samples is gated according to its internal control (small panel represented in the top left part of the plot). Error bars± s.e.m. Two experiments are shown. Assay replicated more than three times in the laboratory. (h) mRNA analysis shows increased CRIPTO and GRP78 expression after coculture. Error bars± s.e.m. Two experiments are shown. Assay replicated more than three times in the laboratory. (i) CM from human osteoblast, generated in the absence of serum, enhances migration of PC-3M-Pro4Luc2 human PCa cells. Error bars ± s.e.m. Two experiments are shown. Assay replicated three times in the laboratory. (j) Coculture of PC-3M-Pro4Luc2_dTomato PCa cells for 48 h with human osteoblast induces a shift to mesenchymal phenotype (decrease in ratio E-cadherin/vimentin and E-cadherin/N-cadherin). Error bars±s.e.m. Two experiments are shown, assay replicated more than three times in the laboratory. *Po0.05, **Po0.01, *** and $$$Po0.001.

Article Snippet: Oncogene (2017) 4739 – 4749 Suppressing CRIPTO expression with siRNAs Human CRIPTO siRNAs (SR304769A, SR304769B, SR304769C) and negative controls (SR30004, non-targeting NT) where purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Isolation, FACS, Labeling, Control, Generated, Migration

Journal: Oncogene

Article Title: CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

doi: 10.1038/onc.2017.87

Figure Lengend Snippet: Figure 3. CRIPTO knockdown causes loss of the stem cell-like phenotype in PCa cells. (a and b) qRT–PCR and Western blot analysis of CRIPTO expression in control (scrambled shRNA, NT) PC-3M-Pro4Luc2 (first lane) and CRIPTO knockdown PC-3M-Pro4Luc2 cells with shCRIPTO no. 1–3, derived from different shRNA constructs. Two bands of, respectively, 25 kDa (Native) and 17 kDa (Core) for CRIPTO are detected as shown previously (see Results). (c) Knockdown of CRIPTO affects the proliferation in PC-3M-Pro4Luc2. Error Bars ± s.e.m. (d) CRIPTO knockdown leads to a decrease in the size of ALDHhigh sub-population in PC-3M-Pro4Luc2 cells. Error Bars ± s.e.m. (e) CRIPTO knockdown affects clonogenic ability and CRIPTO overexpression is capable of reversing this phenotype in PC-3M-Pro4Luc2. Error bars ± s.e.m. *,$,#Po0.05; **,$$Po0.01; ***Po0.001.

Article Snippet: Oncogene (2017) 4739 – 4749 Suppressing CRIPTO expression with siRNAs Human CRIPTO siRNAs (SR304769A, SR304769B, SR304769C) and negative controls (SR30004, non-targeting NT) where purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Control, shRNA, Derivative Assay, Construct, Over Expression

Journal: Oncogene

Article Title: CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

doi: 10.1038/onc.2017.87

Figure Lengend Snippet: Figure 4. CRIPTO overexpression promotes PCa cell migration and rescues effects of CRIPTO knockdown. (a and b) CRIPTO Knockdown significantly reduces cell migration in PC-3M-Pro4Luc2 and C4-2B cells and CRIPTO overexpression is capable of rescuing completely (PC-3M- Pro4Luc2) or partially (C4-2B) the phenotype. Quantification is shown in (c and d) Error bars ±s.e.m. (e) mRNA analysis in PC-3M-Pro4Luc2 cells and C4-2B (f) after 48 h of CRIPTO overexpression shows decrease in E-cadherin/vimentin ratio. Error bars ± s.e.m. $Po0.05, **Po0.01 and ***Po0.001.

Article Snippet: Oncogene (2017) 4739 – 4749 Suppressing CRIPTO expression with siRNAs Human CRIPTO siRNAs (SR304769A, SR304769B, SR304769C) and negative controls (SR30004, non-targeting NT) where purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Over Expression, Migration, Knockdown

Journal: Oncogene

Article Title: CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

doi: 10.1038/onc.2017.87

Figure Lengend Snippet: Figure 5. CRIPTO knockdown reduces invasion and tumor growth of human PCa cells in vivo. (a) PC-3M-Pro4Luc2_dTomato human PCa cells with scrambled shRNA control (NT) and shCRIPTO no. 2 shRNAs were injected into the duct of Cuvier to monitor extravasation and formation of distant growth in vivo (30 embryos injected per group). (b) CRIPTO knockdown reduces whole-body tumor burden at 4 dpi (days post injection). Error bars ± s.e.m. (c) CRIPTO knockdown reduces the number of extravasated cells at 1 and 4 dpi at the caudal hematopoietic tissue. Error bars ± s.e.m. **Po0.01 and ***Po0.001.

Article Snippet: Oncogene (2017) 4739 – 4749 Suppressing CRIPTO expression with siRNAs Human CRIPTO siRNAs (SR304769A, SR304769B, SR304769C) and negative controls (SR30004, non-targeting NT) where purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Knockdown, In Vivo, shRNA, Control, Injection

Journal: Oncogene

Article Title: CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

doi: 10.1038/onc.2017.87

Figure Lengend Snippet: Figure 6. CRIPTO knockdown inhibits bone metastasis of human PCa cells in vivo. (a) PC-3M-Pro4Luc2 human PCa cells with CRIPTO knockdown (shCRIPTO no. 2) and shRNA scrambled control (NT) injected in the left ventricle of nude mice. Formation of distant growth was monitored weekly with bioluminescent imaging (BLI) measurements. Images are representative of six animals for CRIPTO knockdown and four animals for NT control. (b) Quantification of BLI measurements. Difference is significant at week 5 (Po0.05 with two-way analysis of variance (ANOVA)). CRIPTO knockdown is represented in red, and NT control is represented in blue. Error bars ± s.e.m. (c) Total number of distant growths per mouse in mice injected with either CRIPTO knockdown (ShCRIPTO no. 2, red) or control (NT, blue) PC-3M-Pro4Luc2 cells (*Po0.05; **Po0,01; with two-way ANOVA). Error bars ± s.e.m. (d) Total number of distant growths (long bones, mandible, spine) per mouse in mice injected with either CRIPTO knockdown (ShCRIPTO no. 2, red) or control (NT, blue) PC-3M-Pro4Luc2 cells (**Po0.01; with two-way ANOVA). Error bars ± s.e.m.

Article Snippet: Oncogene (2017) 4739 – 4749 Suppressing CRIPTO expression with siRNAs Human CRIPTO siRNAs (SR304769A, SR304769B, SR304769C) and negative controls (SR30004, non-targeting NT) where purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Knockdown, In Vivo, shRNA, Control, Injection, Imaging

Journal: Oncogene

Article Title: CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

doi: 10.1038/onc.2017.87

Figure Lengend Snippet: Figure 7. Schematic representation of the proposed role of CRIPTO in the metastatic cascade in human prostate cancer. CRIPTO and GRP78 influence cell proliferation and are highly expressed in highly metastatic stem/progenitor-like cells (ALDHhigh). CRIPTO and GRP78 knockdown impairs cell migration. CRIPTO- and GRP78-expressing cells are better adapted to surviving in the circulation and of forming distant metastases in zebrafish and mouse xenograft models.

Article Snippet: Oncogene (2017) 4739 – 4749 Suppressing CRIPTO expression with siRNAs Human CRIPTO siRNAs (SR304769A, SR304769B, SR304769C) and negative controls (SR30004, non-targeting NT) where purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Knockdown, Migration, Expressing