Figure EV2. Upon stress, inhibition of translation prevents the recruitment of HSPA1A and HSPA8 inside nucleoli (related to Fig 2).

1. Staining of newly synthesized RNA with 5‐ethynyl uridine (EU, 200 μM) alone or with actinomycin D (Act. D; 4 μM) for 4 h, followed by click chemistry. Nucleic acid is stained with DAPI.
2. DRiP labeling in HeLa cells treated with OP‐puro (25 μM) alone or combined with Act. D (4 μM) for 1 h.
3. HeLa cells were either left untreated (Control) or treated as follows: HS at 42°C for 3 h (HS), alone or combined with CHX (50 μg/ml) or Act. D (3 μM); single CHX or Act. D treatment for 3 h; transcriptional stress for 6 h (MG132 10 μM with Act. D 4 μM) (Audas et al, 2016), alone or combined with CHX (50 μg/ml). After cellular treatment, total RNA was extracted and mRNA levels of HSPA1A were measured by RT–qPCR (RPL0 was used for normalization). Statistical significance via one‐way ANOVA; n = 3, ± s.e.m.
4. GFP‐NCL HeLa Kyoto cells were left untreated or exposed to HS at 42°C for 2 h alone or with CHX; as control, cells were also treated with CHX alone for 2 h (50 μg/ml). Cells were then fixed and stained for HSPA1A and DAPI.
5. Staining of HSPA1A and HSPA8 in GFP‐NCL HeLa Kyoto cells treated with Act. D (3 μM) alone or combined with HS at 42°C for 2 h.
6. Staining of HSPA1A, HSPA8, and DRiPs in HeLa cells subjected to transcriptional stress (MG132 10 μM with Act. D 4 μM) alone or concomitant to translation inhibition (CHX 50 μg/ml) for 6 h. OP‐puro (25 μM) was added during the last 45 min of treatment.

Data information: (A, B, D‐F): Scale bars: 10 μm.