2.2. Cell Culture and Transfection
Human embryonic kidney cells (HEK293, ATCC, Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, St. Louis, MI, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA, TFS) and 1% antibiotic–antimycotic solution (100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, TFS). Rat adrenal pheochromocytoma (PC12) cells (Sigma) were grown in RPMI (Roswell Park Memorial Institute) 1640 (TFS) supplemented with 10% horse serum, 5% FBS, and 1% antibiotic–antimycotic solution on dishes coated with 50 µg/mL collagen type I (Merck, Kenilworth, NJ, USA). Human glioblastoma cells (U251) were cultivated in RPMI 1640 cell culture medium supplemented with 5% FBS and 1% antibiotic–antimycotic solution. All cell lines were grown under standard conditions in humidified atmosphere at 37 °C, with 5% CO2. Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
2.5. Stimulation and Immunoblot
HEK293 cells were seeded on 6-well dishes (1 × 106
per dish, Corning, NY, USA) and transfected with either 2 µg opto-FGFR1 or 2 µg FGFR1–eGFP [19
]. Six hours after transfection, the medium was replaced with reduced serum medium (0.5% FBS) and the cells were incubated overnight. One day after transfection, opto-FGFR1-transfected cells were stimulated for 5 min by 2.5 µW/mm2
blue light in a reptile egg incubator (PT2499, Exo Terra, Hagen, Holm, Germany) equipped with 300 light-emitting diodes. The blue light intensity was measured with a digital power meter connected to the Microscope Power Sensor Head (Thorlabs, Newton, NJ, USA). Cells that were kept in the dark (wrapped in aluminum foil) in the same incubator served as controls. FGFR1–eGFP-transfected cells were stimulated by treating them with 100 ng/mL FGF2 (Stemcell, Vancouver, BC, Canada) and 1 µg/mL heparin sulfate (HepS, Sigma) for 5 min.
The lysis of light-stimulated cells was performed in the incubator under blue light. The dark samples were lysed under non-stimulating red light. Cells were harvested in 50 µL lysis buffer on ice, sonicated and centrifuged (13,000 rpm, 20 min, 4 °C). To prevent ERK signaling, 50 µM MAPK kinase inhibitor (PD98059; Sigma) was added to the cells 2 h prior to light activation or FGF2 stimulation. For the measurement of ERK kinetics, the ligand was washed out two times with PBS and lysates made after 5, 15 and 30 min. In the case of opto-FGFR1-transfected cells, the lysates were obtained after 5, 15 and 30 min of dark period. A total of 30 µg protein per lane was separated by SDS-PAGE and transferred to Immobilon-FL PVDF membranes (Merck, Kenilworth, NJ, USA). Blots were incubated with primary antibodies in Odyssey Blocking Buffer (LI-COR) overnight at 4 °C (pFGFR1, #3476, 1:1000; ERK1/2, #9107, 1:1000; pERK1/2, #9101, 1:2000; AKT, #2920, 1:1000; pAKT, #4060, 1:2000; pPLCγ1, #2821, 1:2000, all from Cell Signaling Technology/CST, Danvers, MA, USA; PLCγ1, #16955, 1:1000, Abcam; GAPDH, sc-32233, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies (IRDye 800CW goat anti-rabbit IgG, #926-32231, and IRDye 680RD goat anti-mouse IgG, #926-68070, both 1:20.000, LI-COR, Lincoln, NE, USA) were applied for 1 h at RT. Signals were recorded with an infrared fluorescence detection system (Odyssey Fc Imaging System).
Long-term light and ligand stimulation for neurite outgrowth assay and immunocytochemistry pheochromocytoma (PC12) cells was used for neuronal differentiation experiments. Cell lines enriched with mV-mem-opto-FGFR1-, mV-cyto-opto-FGFR1- or mV-nucl-opto-FGFR1-transfected cells were obtained as follows. Cells with a low passage number (<P10) were seeded on 6-well plates (BD) and transiently transfected with 1 µg of plasmids. After 24 h, the culture medium was replaced by a medium containing 300 µg/mL Geneticin (G418, TFS), which was changed every other day. When sufficient numbers of resistant cell colonies were observed, the cells were triturated with fire-polished Pasteur pipettes for 10 min and then transferred to collagen-coated culture dishes. For microscopic analysis, cells were plated on 35-mm diameter collagen-coated imaging dishes (IBIDI). PC12 cells were also transiently transfected with 1 µg FGFR1–eGFP. After 6 h, cells were cultured in RPMI 1640 supplemented with 10% horse serum, 5% FBS, and 1% antibiotic–antimycotic solution overnight followed by a starvation medium supplemented with 2 mM L-glutamin, 1% antibiotic–antimycotic solution, and N2 supplement (100×, TFS) for 2 h. FGFR1–eGFP-transfected cells and naive PC12 cells were then treated for 48 h with 100 ng/mL FGF2 + 1 µg/mL HepS and with 100 ng/mL nerve growth factor (NGF), respectively. For light stimulation, the dishes were placed in a humidified atmosphere at 37 °C with 5% CO2 in an incubator with light-emitting diodes as described above. The blue light intensity was measured with a digital power meter and set to 2.5 μW/mm2. The control cells were kept in the dark in the same incubator during stimulation. A repetitive 5 min ON and 55 min OFF cycle was used for stimulation over 48 h.
Cells were then fixed with 4% PFA for 15 min and their neurites were visualized by immunocytochemistry. Neuron-specific class III beta-tubulin (1:1000, 1 h at RT; R&D system) and Alexa 568 goat anti-mouse IgG (1:1000, 2 h at RT; TFS) were applied as the primary and secondary antibody, respectively. Inverted fluorescence microscopy (AxioVert, Zeiss, Oberkochen, Germany) at 40× magnification was used for imaging. Neurite outgrowth was defined if cells exhibited at least one process of more than one cell body diameter length.
For immunocytochemistry, PC12 cells enriched with mV-opto-FGFR1-transfected cells were used. Following serum starvation, light stimulation and fixation, cells were incubated overnight with primary antibodies (pERK1/2, #9101, 1:400, CST; pAKT, #4060, 1:400, CST) followed by incubation at RT for 2 h with Alexa 546 goat anti-rabbit IgG (1:1000, TFS). SP8 confocal laser scanning microscopy (Leica) was used for cell imaging. Controls included the omission of primary antibodies (negative) and stimulation of cells with NGF (positive).