Phase-Contrast Microscopy, Immunofluorescence, DNA Synthesis, WST-1, and Cyto 3D Live–Dead Assays

PC12 (3 × 10⁴) cells were made quiescent for 24 h and embedded in 250 μl of phenol red-free growth factor-reduced Matrigel (10 mg/ml; BD Biosciences, San Jose, CA, United States). Conditioned medium (CM) derived by TNBC cells unchallenged or challenged for 10 days with anti-NGF neutralizing antibody (1,600 pg/ml) was collected and added to PC12 cells. After 6 days, different fields were analyzed using a Leica DMIRB (Leica, Wetzlar, Germany) microscope equipped with C-Plan ×40 or HCX PL Fluotar ×63 objective (Leica). Images were captured using a DFC 450C camera (Leica). TNBC cells on coverslips were made quiescent and after 72 h were rinsed with phosphate-buffered saline (PBS), fixed for 10 min with paraformaldehyde (4%, w/v, in PBS; Merck, Saint Louis, MO, United States), permeabilized for 10 min with Tween (0.1%, v/v, in PBS; Bio-Rad, Hercules, CA, United States), and incubated for 1 h with PBS containing FBS (1%, vol/vol). Cells on coverslips were then incubated with the anti-NGF (1:100, ab6199; Abcam, Cambridge, United Kingdom) antibody overnight at 4°C. After extensive washings in PBS, the coverslips were incubated for 1 h at 37°C with diluted (1:200 in PBS containing 0.01% BSA) fluorescein-conjugated AffiniPure anti-rabbit immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories, West Grove, PA, United States). When indicated in the figures, the nuclei were stained for 5 min with Hoechst 33258 (1 μg/ml; Merck) and the plasma membrane for 10 min with red fluorescent Alexa Fluor^® 594 wheat germ agglutinin (WGA; 5 μg/ml) (Molecular Probes, Invitrogen Ltd., Paisley, United Kingdom). The number of cells positive for NGF (NGF-positive cells) was determined using the formula: percentage of NGF-positive cells = (No. of NGF or pro-NGF-positive cells/No. of total cells) × 100. DNA synthesis was analyzed by BrdU incorporation. To this end, quiescent cells on coverslips were left unchallenged or challenged with NGF in the absence or presence of the indicated compounds for 18 h. After in vivo pulse with 100 μM BrdU (Sigma-Aldrich, St. Louis, MO, United States), BrdU incorporation into the newly synthesized DNA was analyzed as reported (Pagano et al., 2004) using a DMLB (Leica, Wetzlar, Germany) fluorescent microscope equipped with HCX PL Apo ×63 oil and HCX PL Fluotar ×100 oil objectives. Images were captured using a DC480 camera (Leica) and acquired using the Leica Suite software. BrdU incorporation was calculated using the formula: percentage of BrdU-positive cells = (No. of BrdU-positive cells/No. of total cells) × 100. Only PC12 cells that, under basal conditions, incorporated <10% BrdU were used in the indicated experiments. WST-1 reagent (Roche) was used to analyze TNBC cell proliferation, as reported (Di Donato et al., 2019). The resulting values were expressed as the fold increase over the basal level. The Cyto3D live–dead assay kit (TheWell Bioscience, North Brunswick, NJ, United States) was used to detect apoptotic TNBC cells. The kit was used according to the manufacturer’s instructions. Dead cells were visualized by using a DMIRB inverted microscope (Leica) equipped with N-Plan ×10 or HCX PL Fluotar ×40 objective (Leica), and the percentage of dead cells was determined using the formula: [No. of propidium iodide (PI)-positive cells/No. of acridine orange (AO)-positive cells] × 100.