Fig. S3

Autophagy promotes TOP1 degradation during replication stress to prevent protein aggregation, related to Figure 3 (A) Immunoblot showing TEX264 and RNF4 depletion in cells used for clonogenic assay. (B) Immunoblot of TOP1 level in total cell extract (n = 4). Treatment with 50 nM CPT was performed for 24 h, and 2 μM MG132 (MG) or 50 nM BAF was added in the last 8 h. Treatment with 1 μM CPT was performed for 3 h with MG132 or BAF. Quantification of normalized TOP1 protein level. One-way ANOVA, SEM represented; UT, untreated. (C) Immunofluorescence of γ-H2AX and 53BP1 foci after 1 h of 50 nM CPT or 1 μM CPT (n = 3). Scale bar, 10 μm. Quantification of positive nuclei for colocalized foci of both γ-H2AX and 53BP1. Two-way ANOVA. Error bar, SD. (D) Method for isolation of aggregated protein by sequential lysis and denaturation step. The aggregates fraction is insoluble in lysis buffer and 1.5% SDS but solubilized in 100% formic acid to be loaded on the gel. Isolation of the aggregates performed after 12 h of treatment with 50 nM CPT or 1 μM CPT (n = 3); UT, untreated. ∗p < 0.05; ns, not significant.