Axiovert 40c

Images of the spheroids were captured by a camera (Canon PowerShot A640, Canon Tokyo, Japan) attached to a phase microscope (Zeiss Axiovert 40C, Zeiss) or, for quantification of the number of nuclei around the spheroids, by a high content imaging system (Opera Phenix, Perkin Elmer, Waltham, MA, USA). Images were taken with a 5× objective in confocal mode. Image analysis was performed with the Harmony (Perkin Elmer) software. The number of nuclei was normalized to the area of analysis around each spheroid to account for variations in size (see Supplementary Figure S2). Statistical difference between control samples of the SFC086 and SFC840 cell lines, both run in triplicates and counting the number of nuclei around at least 18 spheroids in each channel, was determined by a two-pair t-test at a level of α = 0.05. Statistical difference between control samples and the number of nuclei around at least six spheroids for each compound treatment was determined by a z-test at a level of α = 0.05.

For each MB sample, diameter distribution and concentration were quantified with a Coulter Counter Multisizer 3 (Beckmann Coulter, Krefeld, Germany). For this, 2 μL of MB solution were mixed with 20 ml of ISOTON® II (Beckman Coulter) and measured in a volumetric mode at room temperature. Wide-area optical microscopy (OM) was carried out with a Zeiss Axiovert 40 C with an LD A-Plan 40×/0.5 Ph2 objective (Carl Zeiss Microscopy GmbH, Jena, Germany) at a MB concentration of 1 × 10⁸ MB mL⁻¹. Absorbance intensity of the MB samples was recorded in the 500–900 nm wavelength range using a TECAN Infinite M200 Pro (TECAN group Ltd, Männedorf, Switzerland), where a 150 μL aliquot of 1 × 10⁹ MB mL⁻¹ solutions was collected in a triplicate.

Intracellular neutral lipid accumulation was analyzed by ORO staining, performed as in previous studies [28 (link),90 (link)]. After 72 h treatments, cells were fixed for 1 h with 10% formalin and stained with 0.3% ORO (O0625, Sigma-Aldrich, Tres-Cantos, Spain) diluted in 36% triethyl phosphate during 2 h. After washing the excessive dye with distilled water and resuspending it in isopropanol, the quantification of the lipid content was calculated as the quotient of the absorbance measured at 490 nm by the one at 630 nm in duplicate 96-wells, using a microplate reader (Tecan Infinite M200, Männedorf, Switzerland). The reading at 630 nm corresponds to cell protein content, which was acquired by using the Coomassie brilliant blue G-250 dye for 1 h after finishing the ORO procedure, and extraction of the stain with propylene glycol during 1 h at 60 °C. Data are presented as the ratio of absorbance value between ORO and Coomassie blue staining (n = 12 independent cultures). The ORO staining effectiveness was assessed with a Zeiss Axiovert 40C (Carl Zeiss Inc., Oberkochen, Germany) inverted research-grade microscope equipped with a Canon EOS 1000D digital camera (magnification 20×).

Angiogenesis µ-slides (IBIDI GmbH, Munich, Germany) were coated with Matrigel (10 μl per well; BD Biosciences, San Diego, CA, USA) and incubated for 30 min at 37 °C. The OEPCs growing on T75 flask in EGM-2 medium were detached, suspended in serum-reduced (1% FBS) EGM-2 medium without VEGF (50 μl; 1 × 10⁵ cells ml⁻¹) and seeded on top of polymerized Matrigel. For the conditions with unconjugated and VEGF-conjugated MPs, the OEPCs were mixed with MPs at a ratio of 1:1 (cell:particle) and the cell-particle suspension was seeded on top of polymerized Matrigel after 5–10 min incubation to promote cell-particle interaction. For soluble VEGF condition, 50 ng ml⁻¹ VEGF was used. Cells were incubated under hypoxia conditions (0.5% O₂), with 5% CO₂. Sprout formation was evaluated by phase contrast microscopy (Zeiss Axiovert 40C, Carl Zeiss) 12 and 60 h after the cell seeding.

Photographic documentation was obtained using a Zeiss Axiovert 40C inverted phase contrast microscope (Carl Zeiss Jena GmbH, Jena, Germany) in a conventional way and suitable lighting to obtain an interference contrast effect. A Nikon Eclipse 600 microscope (Nikon, Kanagawa, Japan) equipped for epifluorescence observation of fluorochromized samples was used.

Wound-healing test was performed according to the previously reported and standardized protocol.^{48 (link), 49 (link)} SN12C and A498 cells were seeded on glass overslips in a 10-am tissue colture dish and grown to confluent cell monolayer. Afterward the coverslips were transferred to a 24-well plate and a small area was then scratched using a 200-μl pipette tip, and the cells were then rinsed with PBS to remove the loosen debris of the cells. Media with FBS 10%, BSA 1%, CXCL12, CXCL11 with and without inhibitors were replaced and the plates were incubated at 37 °C and 5% CO₂. The distance between two layers of cells that was scratched by pipette tip was then inspected microscopically at 0, 24, 48 and 72 h, respectively. As the cells migrate to fill the scratched area, images were captured by a digital camera (Canon) attached to microscope (Zeiss axiovert 40 C, Carl Zeiss MicroImaging, Inc., Jena, Germany). The experiments were performed in triplicate.
Actin staining was performed as before.^{50 (link), 51 (link)} Briefly after the wound-healing assay, cells were fixed in 3% paraformaldehyde and permeabilized in 0.2% Triton after extensive washing in PBS. Phalloidin TexRed (Sigma) was used to stain F actin for 45 min in a dark chamber. After PBS washing, coverslips were mounted, analyzed and acquired using confocal microscope (Zeiss LSM 540).

OEPCs were transfected at 40–60% confluency using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s protocols. Inhibition of miR-17 was achieved by transfection of cells with 50 nM miRIDIAN Hairpin Inhibitor (Dharmacon Inc., Lafayette, CO, USA). 50 nM anti-miR negative control from Ambion was used as control in all transfection experiments. siRNA-mediated silencing of ZNF652 and CDKN1A was obtained using 40 nM ON-TARGETplus siRNAs (Dharmacon Inc.) against these targets. After 2 days of transfection, cells in 96-well plates were washed and the cell culture medium was replaced by EBM-2 and cells were incubated under normoxia and hypoxia conditions (0.1% O₂), with 5% CO₂. After 48 h, survival assay (Presto Blue Cell Viability Reagent; Thermo Fisher Scientific) was performed according to manufacturer’s instructions. For Matrigel assay, the cells were detached after transfection, suspended in serum-reduced (1% FBS) EGM-2 medium without VEGF (50 μl; 2 × 10⁵ cells ml⁻¹) and seeded on top of polymerized Matrigel. Cells were incubated under normoxia and hypoxia conditions (0.1% O₂), with 5% CO₂. Sprout formation was evaluated by phase contrast microscopy (Zeiss Axiovert 40C, Carl Zeiss) or INCELL Analyzer 2200 (GE Healthcare) 48 h after the cell seeding.