Alexa fluor 594 and alexa fluor 488 conjugated secondary antibodies

Immunocytochemical staining for vimentin (3.7 μL/mL; ab92547; Abcam, Cambridge, MA) and cytokeratin-18 (CK-18) (1 μL/mL; ab668; Abcam) were performed after the scratch assay, and after 6 days of exposure to the treatment conditions described above (de Castro Silva et al., 2020 (link); Omere et al., 2020 (link); Richardson and Menon, 2018 (link); Richardson et al., 2020 (link); Tantengco et al., 2020 (link)). The dilution factor for primary antibodies was 1:300 for vimentin and 1:500 for CK-18. After each time point, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X, and blocked with 3% bovine serum albumin in PBS before incubation with primary antibodies overnight at 4°C. This protocol is adequate to remove non-specific binding of primary antibodies in our system, based on our previous studies (Richardson and Menon, 2018 (link); Richardson et al., 2020 (link)). After washing with PBS, slides were incubated with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Life Technologies, Carlsbad, CA) and diluted 1:400 in PBS for 1 h in the dark. Slides were washed with PBS, treated with NucBlue Fixed Cell Stains ReadyProbes Reagent (R37606; Thermo Fisher Scientific, Watham, MA), and then mounted using Mowiol 4 to 88 mounting medium (475904–100GM-M; Sigma-Aldrich, Inc.).

NCI-H1563 cells were treated with 2 μg ml⁻¹ doxycycline (dox) for 24 h to induce A3H Hap I-Flag and fixed with 100% cold methanol for 10 min. Cells were permeabilized with 100% cold acetone for 1 min and 0.5% Triton X-100 for 10 min. Anti-Flag and anti-γH2AX antibodies (Invitrogen) in 3% bovine serum albumin in 4 saline–sodium citrate buffer were incubated 1 h. Primary×antibodies were detected with Alexafluor-594- and Alexafluor-488-conjugated secondary antibodies (Invitrogen). Nuclei were stained with DAPI and cells were imaged using the Zeiss LSM700 system. Data were compiled from 373 cells from three trials.