Dm 14000b confocal microscope

The cells were seeded onto coverslips in a six‐well plate. Then, they were fixed with 4% paraformaldehyde (w/v) for 30 min. Coverslips with glioma cells were then washed with PBS for 15 min and permeabilized with 0.2% (w/v) Triton X‐100 in PBS for 5 min. Then, they were blocked with PBS containing 1% bovine serum albumin (BSA) for 30 min. Coverslips were subsequently incubated with the primary antibodies against cytochrome c, p65, and p50 diluted in PBS containing 10% BSA overnight. Coverslips were washed with PBS for three times, and then, fluorescein isothiocyanate‐ and rhodamine‐conjugated secondary antibodies were added in 1% blocking solutions and incubated for 1 h. After three times washes with PBS, coverslips with glioma cells were stained with DAPI (Beyotime). Samples were examined using a Leica DM 14000B confocal microscope.

HUVECs were seeded onto coverslips in a 6-well plate. The cells were treated with or without 50 μM CS-6 for 1h and then were stimulated with 50 ng/mL VEGF for 15min. After that, the cells were fixed in 4% paraformaldehyde for 20 min, and then permeabilized with 0.2% Triton X-100 for 5 min at room temperature. Actin filaments were stained by phalloidin-FITC for 20 min, and nuclei were detected by DAPI. For phosphorcofilin immunofluorescence, cells were blocked with 5% BSA in PBS for 1 h. After washing three times with PBS, cells were incubated with antibody against phospho-cofilin overnight at 4°C, and then with secondary antibodies for another 1 h at room temperature. Next, cell nuclei were stained by DAPI for 5 min. The slides were examined with a Leica DM 14000B confocal microscope.

IF staining was performed in cells cultured in chamber slides. After dopamine treatment, the cells were washed in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). The samples were then permeabilized with 0.2% TritonX-100 for 5 min before being blocked with 10% bovine serum albumin (BSA) in PBS for 30 min. Antibodies against cytochrome c, p65 and p50 in 1% blocking solution were subsequently added to the samples, which were incubated overnight at 4°C. Following three 10-min washes with PBS, the cells were treated with fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies in 1% blocking solution and incubated for 1 hr. Then, the stained samples were mounted with 4’, 6-diamidino-2-phenylindole (DAPI)-containing Vectashield solution (Vector Laboratories Inc.) to counterstain the cell nuclei. After five additional 5-min washes, the samples were examined with a Leica DM 14000B confocal microscope.

Briefly, cells on chamber slides were fixed with 4% paraformaldehyde and permeabilized with 0.2% TritonX-100. The samples were probed with specific antibodies against AHNAK (Santa Cruz) and p-IGF-1R (Cell Signaling Technology) at 4 °C for 12 h and then incubated with FITC- and TRITC-labeled secondary antibodies (1:200 dilution) for 1 h. After each step, cells were washed two times using PBS. The cell nuclei were stained with DAPI for 3 min, and a Leica DM14000B confocal microscope was used to capture images.