H1944 cells, seeded onto coverslip, were treated with increasing concentrations of LL6 (0, 1, 2.5 μM) for 24 h. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, and then permeabilized with 0.3% Triton X-100 for 15 min at room temperature. After washing cells with PBS, the cells were incubated with blocking solution [3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20] for 1 h at room temperature. Cells were incubated with primary antibodies (1:200 dilution) at 4 ºC overnight. Cells were washed several times with PBS and incubated with fluorochrome-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Cells were washed multiple times with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted with mounting solution (Dako, Glostrup, Denmark) and then observed under a fluorescence microscope (Zeiss Axio Observer Z1, Carl Zeiss AG, Oberkochen, Germany).

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