Fig. S6

TEX264 with p97 and SUMO acts as the receptor for TOP1cc degradation by autophagy, related to Figure 6 (A) Immunofluorescence of TOP1cc foci in WT cells, TEX264KO, or TEX264KO complemented with TEX264WT after 2 h of 50 nM CPT, followed by 2 h of recovery. Quantification TOP1cc foci per nuclei, one experiment shown (n = 3). Two-way ANOVA. Error bar, SD. (B) Quantification of a representative clonogenic assay in WT, TEX264KO or TEX264KO complemented with TEX264WT after 8 h of CPT treatment (n = 3). Error bar represents mean ± SEM. Two-way ANOVA. (C) Co-immunoprecipitation of TEX264-V5 either WT or TBS∗, LIR∗ or SHP∗ mutants to confirm the loss of interaction with respective TEX264-binding partners. (D) Lysate of cells expressing TEX264 WT or P∗ mutant. The antibody used to detect phosphorylation specifically recognizes TEX264 phosphorylated on serines 271 and 272. (E) LysoIP was performed after 5 h of treatment with 50 nM CPT and the inhibitor of SUMO E1 activating enzyme ML792. siRNA targeting RNF4 and PIAS4. All conditions were treated with 50 nM BAF (n = 3). (F) LysoIP performed after 5 h of treatment with 50 nM CPT and 10 μM of p97 inhibitor CB-5083 (CB). All conditions were treated with 50 nM BAF (n = 3). (G) Immunofluorescence of TOP1cc foci after 2 h of 50 nM CPT. Recovery was performed for 2 h in media or with the p97 inhibitor CB-5083 (CB). Scale bar, 10 μm. Quantification of positive nuclei for TOP1cc foci (n = 3). Two-way ANOVA. Error bar, SD. (H) LysoIP performed after 3 h of treatment with 50 nM CPT, depletion of SPRTN achieved using siRNA against SPRTN; control siLUC (n = 3). All conditions were treated with 50 nM BAF. ∗p < 0.05; ∗∗∗p < 0.0005; ns, not significant.