Dyskerin

Cells were seeded on glass coverslips placed in 6-well plates and treated for 96 h with Dox. They were then fixed with 3.7% paraformaldehyde for 10 min, permeabilized in 100% ice-cold methanol for 10 min at −20 °C and finally blocked with DPBS supplemented with 3% bovine serum albumin (BSA) for 30 min. After each step, cells were rinsed in DPBS and incubated with primary antibodies overnight at 4 °C, following which the cells were washed in DPBS and incubated with secondary antibodies at RT for 1 h on the next day. Glass coverslips were counterstained with DAPI, mounted on glass slides and examined under a Zeiss confocal fluorescence microscope (Zeiss LSM 700). The primary antibodies used were the following: Calreticulin (D3E6) XP (Cell Signaling Technology (CST), Heidelberg, Germany, 12238; 1:250), Dyskerin (Santa Cruz Biotechnology, Dallas, TX, USA, sc-373956; 1:1000), GRP78/BiP (Thermo Fisher Scientific, Waltham, MA, USA, PA5-11418; 1:500) and LC3 (Abcam, CA, USA, ab51520; 1:3000). The secondary antibodies used were: Cy3-AffiniPure Goat Anti-Rabbit IgG (H + L) (Invitrogen, Van Allen Way Carlsbad, CA, USA, A10520; 1:500) and Alexa Fluor 488 donkey anti mouse IgG (H + L) (Invitrogen, Van Allen Way Carlsbad, CA, USA, A21202; 1:1000).

Lysates were prepared using RIPA buffer (Sigma) containing protease inhibitor (Roche). The protein concentration of the cell lysates was determined by bicinchoninic acid assay (BCA, Pierce, Rockford, IL), stored at –20°C. 200 µg of total protein were immune precipitated by µMACS™ Protein A/G MicroBeads MultiMACS™ Protein A/G Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and performed according to the manufacturer’s instructions. The following immunoblot analyses were performed as described above. Antibodies for immunoprecipitation: ILK (4 µg per immunoprecipitation; SantaCruz), c-Fos (2 µg per immunoprecipitation; SantaCruz). Primary antibodies: ILK (1:200; SantaCruz), c-Fos (1∶200; SantaCruz), kindlin-1 (1∶500; polyclonal rabbit; Abcam), dyskerin (1∶200; SantaCruz)

Genotypes of yeast strains and plasmids are listed in Appendix Tables S2 and S3. All chemicals used in this study were purchased analytical grade from either Sigma‐Aldrich or Carl Roth, unless stated otherwise. Drop Out Mix for yeast synthetic medium was from US Biological Life Sciences (D9543‐01). 5‐FOA was bought from Cayman Chemicals (17318). Protein‐G coupled Dynabeads™ (10004D) and streptavidin‐coated Dynabeads™ MyOne™ Streptavidin C1 (65001) were from Thermo Fisher. Secondary antibodies against rabbit (7074S) and mouse IgG (7076S) coupled to HRP were purchased from Cell Signaling. Primary antibodies were as follows: H3K56ac (Active Motif – 39281), G6PDH (Sigma – A9521) and c‐Myc (9e10, Sigma – MABE282), FLAG (Sigma – F3165), H2AQ105me from (Tessarz et al, 2014 (link)), H2A (Abcam – ab13923), H3 (Abcam – ab1791), Utp11L (GeneTex GTX115929), Dyskerin (Santa Cruz sc‐373956) and human Nhp2 (Proteintech 15128‐1‐AP). ECL solution was from Promega (W1001). T4 Polynucleotide kinase was from NEB (M0201) and Probe Quant G‐50 microcolumns from GE Healthcare (28‐9034‐08). γ‐(³²P)‐ATP was purchased from Perkin Elmer (BLU002Z250UC).