Alex fluor 647

Human CD3+ cells were isolated and stimulated as described above, for 2 days. The day before co-culture, NCI-H2023 tumor cells were plated in 96-well 190 μM glass bottom plates (Greiner). The following day, tumor cells were treated for 30 min with Cell Tracker Orange (Invitrogen), HCS Nuclear Mask Blue (Invitrogen), and for 1 h with 150 μg of the human PD1-Fc-OX40L ARC previously labeled with Alex Fluor 647 (Invitrogen). Media was then removed and activated human T cells were seeded on the tumor cells at a ratio of 8:1 (T cell: tumor cell), along with CellEvent Caspase 3/7 Green Detection Reagent (Invitrogen). Images were then acquired over the course of 6 h at a 40X magnification on the GE IN Cell Analyze 2500HS System.

The cells in the culture glasses were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 and 10% bovine serum albumin (BSA) for 45 min. Next, the cells were incubated with specific primary antibodies overnight at 4 °C and then incubated with secondary antibodies in 10% BSA containing 0.1% Triton X-100. Nuclei were counterstained with DAPI for 5 min. Images were acquired with a fluorescence microscope (Olympus, IX73, and Japan). Two independent experiments are performed. The primary anti-NY-ESO-1 markers were from Cell Signaling Technology, and the secondary antibody with fluorescence Alex Fluor™ 488 and Alex Fluor™ 647 were from Invitrogen (Massachusetts, USA).

Rabbit anti-TCOF1/treacle was purchased from Proteintech (Cat #11003-1-AP) and used for immunofluorescence (IF; 1:100) and immunoblotting (IB; 1:2000). Mouse anti-GFP (Sigma-Aldrich, Cat #11814460001 ROCHE; 1:2000), anti-β-tubulin (Sigma, Cat # T8328; 1:2000), and anti-β-actin (Abcam, Cat# Ab3280; 1:2000) were used for IB. Mouse anti-HeV M (Generated at AAHL; 1:10)^{42 (link)} was used for IF^{25 (link)}. Anti-FBL (Abcam, Cat #Ab4566), anti-NCL (CST, Cat#14574), anti-UBF1/2 (gift from Prof. Ross Hannan (Australian National University), in-house generated antibody^{43 (link)}, 1:100) and anti-Phospho-Histone H2A.X (Ser139) (γH2AX; CST, Cat#2577; 1:800) were used for IF and/or immunoblotting. Secondary antibodies diluted 1:1000 were used for IF (goat anti-rabbit Alexa Fluor 568 (Cat #A-11011), goat anti-rabbit Alex Fluor 647 (Cat #A-21245)) were purchased from Thermo Fisher Scientific. Secondary antibodies diluted 1:10,000 were used for IB (goat anti-rabbit (Cat #AP307P) and goat anti-mouse (Cat #AP308P) IgG horse radish peroxidase (HRP)-conjugated-antibodies) were purchased from Merck. rRNA synthesis was inhibited by treating cells with Actinomycin D (ActD) (Thermo Fisher Scientific, Cat #11805017) for 1 h at 500 ng/mL. DNA damage was induced by treating cells with 50 μM etoposide for 3 h.