Axiocam 506 mono system

Cells were grown on coverslips for 24 h. HEK293T were transfected with AATK wt-EYFP, AATK KD-EYFP or empty control, and AATK wt or AATK KD expression was induced with doxycycline in the TREx clone pools. After 24 h, cells were washed, fixed in 3.7% formaldehyde–PBS solution, permeabilized with 0.5% Triton X-100–PBS solution, and blocked with 1% BSA-PBS solution. For HEK293T, incubation with TP53Ser366 (#A8053 Assay Biotech) at 1:50 and subsequent incubation with anti-rabbit Alexa568 was performed. For the TREx clone pools, simultaneous incubation with primary antibodies against TP53Ser366 (#A8053 Assay Biotech) at 1:50 and Anti-FLAG M2 (#F1804 Sigma-Aldrich) at 1:100 in 1% BSA-PBS solution, subsequent simultaneous incubation with secondary fluorescent antibodies (anti-mouse Alexa568 and anti-rabbit Alexa488) were performed. Thereafter, cells were stained with DAPI (Invitrogen) and mounted with Mowiol. Images were acquired using an Axio Observer.Z1 inverted microscope (Carl Zeiss) equipped with Zeiss Zen 3.1 (blue edition) software and the Axiocam 506 mono system (Carl Zeiss). Processing of the images was performed with Fiji/ImageJ (version 1.51n). Bean plots were generated via http://shiny.chemgrid.org/boxplotr/.

IF stainings were performed as described in Reference [84 (link)], with a few adjustments. Briefly, HeLa Kyoto cells were cultured on coverslips and fixed with 3% paraformaldehyde in PBS. After three PBS washes, cells were permeabilized with PBS containing 0.5% Triton X-100, followed by blocking with 1% bovine serum albumin (BSA) in PBS. For target protein detection, coverslips were stepwise incubated with primary and then secondary Alexa Fluor–conjugated antibody. After washing, DNA was visualized by using 10 µg/mL Hoechst in PBS. Coverslips were mounted in Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL, USA). Images were acquired with an Axio Observer.Z1 inverted microscope (Carl Zeiss, Oberkochen, Germany) with Axiocam 506 mono system. Image processing was performed with Zeiss Zen 3.1 (blue edition) software and ImageJ (version 1.51n).