Additional file 2: of The phospholipase DDHD1 as a new target in colorectal cancer therapy

Figure S1. DDHD1 silencing. To evaluate DDHD1 silencing a. Real-time PCR and b. Western blot analysis were performed on SW480, HCT116, HS5 and HUVEC transfected for 48 or 72 h with scrambled siRNA or DDHD1 siRNA. (TIFF 6629 kb

SEM characterization of cell seeded scaffolds.

<p>Detailed analysis of cell morphology was assessed by scanning electron microscopy. A) Control at day 7. B) HA/Mgn 95/5 at day 7. C) HA/Mgn 90/10 at day 7. D) HA/Mgn 50/50 at day 7. E) Control at day 14 with applied magnetic field. F) HA/Mgn 90/10 at day 14 with applied magnetic field. G) HA/Mgn 95/5 at day 7, vertical overlapping profile of 3 consecutive images. H) Control at day 7. I, J) HA/Mgn 90/10 at day 7. K) HA/Mgn 95/5 at day 7. Scale bars: A–D) 150 µm, E, F) 10 µm, G) 500 µm, H, I) 50 µm, J) 50 µm, K) 20 µm.</p

Analysis of cell morphology.

<p>Cell morphology was analysed by actin staining (n = 3). Actin is shown in green, DAPI in blue. A) Control at day 14. B) HA/Mgn 90/10 at day 14. C) Control at day 21 with applied magnetic field. D) HA/Mgn 90/10 at day 21 with applied magnetic field. Scale bars: A, B) 250 µm. C, D) 100 µm.</p

Additional file 3: of The phospholipase DDHD1 as a new target in colorectal cancer therapy

Table S1. Data from SWATH-MS Gene Ontology analysis. (XLSX 740 kb

Cell proliferation assay.

<p>The Picogreen DNA content assay was performed on cultures of osteoblast-like cells seeded on different HA/Mgn scaffolds at 7, 14 and 21 days of culture, either in the presence or absence of a magnetic field (MF) (n = 5). HA porous scaffold is used as control group. * p≤0.05.</p

Additional file 4: of The phospholipase DDHD1 as a new target in colorectal cancer therapy

Figure S2. Effects of DDHD1-expressing cells conditioned medium on DDHD1-silenced cell growth. Cell viability was measured by MTT assay on DDHD1-silenced SW480 cells in the presence of the conditioned medium (CM) of mock cells and DDHD1 overexpressing cells. (TIFF 3275 kb

Alkaline phosphatase activity assay.

<p>AP activity was measured on different HA/Mgn scaffolds seeded with human osteoblast-like cells at 7, 14 and 21 days, either in the presence or absence of a magnetic field (MF) (n = 5). Results are normalized to control.</p

Histological evaluation of the <i>in vivo</i> implanted scaffolds.

<p>Toluidine Blue, Acid Fucsin and Fast Green staining shows similar histocompatibility for both scaffolds 4 weeks after implantation (n = 6). A, B) Control, C, D) HA/Mgn 90/10. Scale bars: A, C) 1 mm, B, D) 500 µm.</p

Analysis of cell viability.

<p>Cell viability was analysed by the Live/Dead assay (n = 3). Live-Dye stains for live cells in green, Propidium Iodide stains for dead cells in red. A) Control at day 7. B) HA/Mgn 95/5 at day 7. C) HA/Mgn 90/10 at day 7. D) HA/Mgn 50/50 at day 7. E) HA/Mgn 95/5 at day 14. F) HA/Mgn 95/5 at day 14. G) HA/Mgn 50/50 at day 14 with applied magnetic field. Scale bars: A–D) 500 µm. E) 250 µm. F, G) 100 µm.</p