96 well plate

The cell cycle distribution was analyzed using a FACSCalibur^TM (BD Biosciences, Heidelberg, Germany), as reported [20 (link)]. Briefly, cells were harvested, washed with PBS, fixed in chilled 70% ethanol at 4 °C for 30 min., treated with 1 mg/mL of RNase A (Sigma-Aldrich, Munich, Germany) and stained with 100 μg/mL of propidium iodide (PI) for 30 min. at 37 °C. DNA content was determined.
Cell proliferation assays were carried out by using Cell Titer-Blue^® Cell Viability Assay (BD Biosciences, Heidelberg, Germany) on treated cells in 96-well plates (Promega, Mannheim, Germany). 20 μL of CellTiter-Blue^® reagent was added to each well and then incubated at 37 °C with 5% CO₂ for 4 h before fluorescence reading while using a Victor 1420 Multilabel Counter (Wallac, Finland), as reported [20 (link)].

Cell proliferation assays were carried out by using Cell Titer-Blue^® Cell Viability Assay on treated cells in 96-well plates (Promega, Mannheim). 20 μl of CellTiter-Blue^® reagent was added to each well and then incubated at 37°C with 5% CO₂ for 3 h before fluorescence reading using a Victor 1420 Multilabel Counter (Wallac, Finland). Cell cycle was analyzed using a FACSCalibur^TM (BD Biosciences), as described [55 (link)]. Briefly, cells were harvested, washed with PBS, fixed in chilled 70% ethanol at 4°C for 30 min, treated with 1 mg/ml of RNase A (Sigma-Aldrich) and stained with 100 μg/ml of propidium iodide (PI) for 30 min at 37°C. DNA content was determined. Early apoptosis (Ann+) and late apoptosis (Ann+PI+) were assessed using Vybrant™ apoptosis assay kit #2 according to the instructions (Molecular Probes, Leiden). Apoptosis was measured with a FACSCalibur^TM (BD Biosciences). The data were analyzed by using the cell cycle analysis software CellQuest (BD Biosciences). The activity of caspase-3/7 was measured in triplicate with Caspase-Glo^® 3/7 Assay as instructed (Promega).

The cell viability was measured using the MTS-based cell viability assay in 96 well plates (Promega, Les Ulis, France). To test FGF-2 dosage effects, HUVEC or HDMEC was plated in full medium for 24 h, then EBM-2 basal medium was added and cells were treated or not with 1nM or 3nM FGF-2 for 72 additional hours. To study the impact of splicesome inhibitors on FGF-2 effects, a similar protocol was used with endothelial cells being treated or not with 3nM FGF-2 in the presence or absence of SRPK1/2 inhibitors SPHINX31 (5μM) or SRPIN340 (10μM). To test the impact of SRPK1 knockdown on cell viability, 0.25 × 10⁶ HDMEC cells/well were plated in 6-well plates in full medium for 24 h, subjected to two rounds of transfection in full medium as described above, then plated in EBM-2 basal medium and treated or not with 3nM FGF-2 for 48 additional hours. Four technical replicates were done per condition. After treatment, cells were washed with PBS 1X. Then, 10 μl of WST-1 reagent was added to each well. Plates were incubated at 37°C for 2 h. After the incubation period, plates were mixed gently on an orbital shaker for 1 min and the absorbance of each sample was measured at 492 nm using Beckman Coulter AD 340s (Fullerton, CA, USA).

SRA-hLECs, mLECs, or different ages of primary hLECs were transfected with pRBGP2 (3x ARE-LUC) containing three ARE sites or its mutant pRBGP4 (3x mut ARE- LUC) plasmids, a kind gift from Dr. Hozumi Motohashi, Japan) [82 (link)] along with Renilla, pRL-TK vector (Promega, Madison, WI, USA) using Neon transfection system (Invitrogen, Waltham, MA, USA). After 14 h, cells were washed and treated with Hyd for 24 h. Luciferase activity was measured using Dual-Glo luciferase assay system with 96-well plate (Promega, Madison, WI, USA) submitted to a microplate reader (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA) [3 (link)].

The SCI1 promoter was amplified in two different fragments, a long one (−1585 to −1) and a small fragment close to the ATG (−538 to −1). Both fragments were inserted in the pGreenII 0800-LUC vector (Hellens et al., 2005 (link)), which has two different luciferase genes: the Renilla luciferase gene used as an internal control of the transient expression and the firefly luciferase gene under the control of the SCI1 promoter sequences in this study (reporter construct). The NAG1 and NtWUS CDS were cloned into the vector pK7WG2 (Karimi et al., 2002 (link)) for protein expression under the 35S promoter control and used as effectors. All gene constructs were sequenced before use. The reporter and effector constructs were introduced separately into A. tumefaciens strain GV3101. Different combinations of reporter and effector constructs were agroinfiltrated in Nicotiana benthamiana leaves for transient expression. The activity assay was performed 3 days after agroinfiltration. Firefly (LUC) and Renilla (REN) luciferases were detected with the dual luciferase assay reagents (Promega, Madison, WI, United States) using Costar^® 96-well plate. The promoter activity was calculated using the LUC/REN ratio. The data were analyzed by Student’s t-test.