Cells were fixed using 3% paraformaldehyde for 10 min after which they were permeabilized using 0.1% Triton X- for 10 min. Primary antibodies were incubated overnight at 4°C. Following 3 washing steps with PBS, cells were incubated with secondary antibody for 1 h at 37 °C at a 1/200 dilution. After three more washing steps with PBS, cells were mounted using glycerine-DABCO.
Stained samples were imaged using a Leica SPE laser scanning confocal microscope (Leica) or a Dmi8 Thunder inverted microscope (Leica). Images were processed and analyzed using FIJI. For the quantification of P-bodies the analyze particles function was used, with a minimum size of 10 pixels, after setting a threshold of 150 to determine EDC4 puncta. Colocalization of YTHDF proteins and P-bodies was quantified by measuring the degree of overlap of the fluorescence signal in the YTHDF channel with the fluorescence signal in the EDC4 channel using Fiji. Per condition, for each of the three independent replicate samples, three random fluorescence images taken with a Leica Dmi8 Thunder inverted microscope (Leica) were used for analysis. Immunofluorescence assays were performed using primary antibodies against YTHDF1 (Proteintech 17479-1-AP, 1/100), YTHDF3 (Proteintech 25537-1-AP, 1/100 and EDC4 (Santa Cruz Biotechnology sc-376382, 1/100).
Stained samples were imaged using a Leica SPE laser scanning confocal microscope (Leica) or a Dmi8 Thunder inverted microscope (Leica). Images were processed and analyzed using FIJI. For the quantification of P-bodies the analyze particles function was used, with a minimum size of 10 pixels, after setting a threshold of 150 to determine EDC4 puncta. Colocalization of YTHDF proteins and P-bodies was quantified by measuring the degree of overlap of the fluorescence signal in the YTHDF channel with the fluorescence signal in the EDC4 channel using Fiji. Per condition, for each of the three independent replicate samples, three random fluorescence images taken with a Leica Dmi8 Thunder inverted microscope (Leica) were used for analysis. Immunofluorescence assays were performed using primary antibodies against YTHDF1 (Proteintech 17479-1-AP, 1/100), YTHDF3 (Proteintech 25537-1-AP, 1/100 and EDC4 (Santa Cruz Biotechnology sc-376382, 1/100).