Triton x 100 0.2

U-937 cells were seeded in ibiTreat µ-Slide 8 Well chambers slide (Ibidi; Germany, Cat#80826) (200 000 cells/well) after being subjected to lentiviral transductions. Cells were fixed with 4% (w/v) paraformaldehyde (Thermo Fisher Scientific; Cat#28906), permeabilized with Triton X-100 0.2% (Sigma-Aldrich; Cat#X100), and incubated with anti-FLAG mouse monoclonal primary antibody (1:500; Merck; Cat#F1804) and Alexa Fluor 488 rabbit-anti-mouse IgG (1:1000; Invitrogen; Cat# A27023) secondary antibody. Cells were washed with PBS between steps and incubated with DAPI (5 min; 1:5000; Invitrogen; D1306) before mounting with ibidi mounting medium (Ibidi; Cat#50001). Fluorescence images were acquired using a Leica-SP8 confocal microscope with a HC PL APO CS2 40/1.10 water objective and analysed using the Bitplane Imaris software (Imaris, Zurich, Switzerland).

Immunofluorescence analysis was performed in H1299 cells fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with Triton X100 0.2% (Sigma-Aldrich) and blocked with 3% BSA (all from Sigma-Aldrich). Cells were stained with anti-Myc-Tag (9E10, Sigma-Aldrich) - for INSL4 analysis - and anti-calreticulin (StressGen Biotechnologies Corp., BC, Canada) antibodies. Goat anti-Mouse IgG (H+L) Alexa Fluor^® 555 conjugate and Goat anti-Rabbit IgG (H+L) Alexa Fluor^® 488 conjugate secondary antibodies were used respectively (Thermo Fisher Scientific, Waltham, MA, USA). DNA was stained with DAPI (Sigma-Aldrich). Cells alternatively stained with a fluorescent phalloidin-FITC conjugate (Sigma-Aldrich) solution in PBS at room temperature for 40 minutes. Phalloidin-FITC conjugate were used as a cytoskeleton marker.
Fluorescence analysis was performed using Zeiss Axioplan (Zeiss, Oberkochen, Germany) fluorescence microscope controlled by Spot-2 cooled camera (Diagnostic Instruments, Sterling Heights, USA). Experiments were performed at least three times and performed each time in triplicate. Images were exported in TIFF, and contrast and brightness were adjusted in Corel Paint Shop Pro X9 and final figures were generated with Adobe Illustrator 6.