Figure 2. REV-ERBs agonist SR9009 inhibits autophagy.

a–b, SR9009 treatment reduces the number of autophagosomes, as shown by immunofluorescence of LC3B, (n=biological independent samples MCF7 (n=6 mock), (n=5 SR9009) and T47D (n=5 mock) (n=4 SR9009) Mann–Whitney test one-tailed MCF7 20µM 24h *P=0.0152, T47D 20µM 48h **P=0.0079; c–d SR9009 induces accumulation of p62 as shown by immunofluorescence; n= biological independent samples MCF7 (n=3 mock), (n=8 SR9009) and T47D (n=5 mock), (n=4 SR9009) Mann–Whitney test one-tailed 48h MCF7 p62 **P=0.0061; 48h T47D **P=0.0079; e, Inhibition of autophagy is confirmed by the immunoblot for p62 (20µM 48h, A375); f–g, Inhibition of autophagy precedes apoptosis induction as shown by immunofluorescence of p62, cleaved Caspase 3 and TUNEL assay; n=biological independent samples, Mann–Whitney test one-tailed, A375 20µM Cl. Casp. 3 48h (n=3) *P=0.0179; Cl. Casp. 3 72h (n=7) ****P<0.0001; Tunel 48h (n=3) *P=0.0179; Tunel 72h (n=7) ****P<0.0001; p62 48h ****P<0.0001 (n=8); p62 72h (n=9) ****P<0.0001; h, Starvation dramatically accelerate the cytotoxic effect of REV-ERB agonist SR9009 (A375, 3 days 20µM, starvation time 24h; i, Overexpression of ULK3 impairs SR9009 induction of apoptosis (MCF-7, 6 days 20µM); j, Overexpression of ULK2 and LKB1 impairs SR9009 induction of apoptosis (A375, 6 days 20µM). k, WST-1 viability assay shows abrogation of apoptosis in ULK2 and LKB1 overexpressing cells (n=biological replicates A375, 6 days 20µM; Mann–Whitney test one-tailed n=12, E.V. Mock vs E.V. 09 ****P<0.0001; ULK2 Mock (n=12) vs ULK2 09 (n=10) ****P<0.0001; n=12 E.V. Mock vs E.V. 09 ****P<0.0001 and LKB1 Mock vs LKB1 09 **P=0.0028. All scale bars 50 µm. All panels three biological independent experiments with similar results. All the data are plotted as mean ± s.e.m. For gel source data, see Supplementary Fig. 1.