Extended Data Fig. 5: PF-670462 increases the ferroptosis sensitivity of tumor cells via zDHHC8-GPX4 axis.
a, The cell viability of HT-1080 cells treated with RSL3 (0.05 μM) in conjunction with or without PF-670462 (10 μM) and DFO (5 μM), Lipro-1 (5 μM), Z-VAD-FMK (10 μM) or NSA (2.5 μM) for 24 h. b, LDH release measurement of HT-1080 cells treated with RSL3 (0.05 μM) in conjunction with or without PF-670462 (10 μM) and Fer-1 (2 μM), DFO (5 μM), Lipro-1 (5 μM), Z-VAD-FMK (10 μM) or NSA (2.5 μM) for 16 h. c, The cell viability of HT-1080 cells treated with the indicated concentrations of ML162 or IKE in conjunction with or without PF-670642 (10 μM) and Fer-1 (2 μM) for 24 h. d, The cell viability of various tumor cells treated with the indicated concentrations of RSL3 with PF-670462 (10 μM) for 24 h. e,f, The cell viability of HT-1080 cells treated with the indicated concentrations of RSL3 with SR-3029 (e) or LH846 (f) for 24 h. g, Immunoblotting analysis of CK1ε and CK1δ in control (shCtrl) or CK1δ/ε-knockdown (shCK1δ/ε) HT-1080 cells. h, The cell viability of shCtrl and shCK1δ/ε HT-1080 cells after treatment with the indicated concentration of RSL3. i, Immunoblotting analysis of thermal stability of zDHHC8-Flag protein by CETSA after the treatment of DMSO, LH846 (10 μM) or PF-670462 (10 μM) in HEK293T cells transfected with plasmid encoding zDHHC8-Flag. j, Immunoblotting analysis of thermal stability of zDHHC8 (NTD)-Flag protein by CETSA after the treatment of DMSO or PF-670462 (10 μM) in HEK293T cells transfected with plasmid encoding zDHHC8 (NTD)-Flag. k, Immunoblotting analysis of thermal stability of zDHHC-Flag protein by CETSA after the treatment of DMSO or PF-670462 (10 μM) in HEK293T cells transfected with plasmid encoding zDHHC5-Flag, zDHHC9-Flag or zDHHC20-Flag. l, Real-time PCR analysis of ZDHHC8 mRNA level in HT-1080 cells treated with the indicated concentration of PF-670462 for 24 h. For a,c-f and h, data are presented as mean ± s.d. of n = 4 biological replicates per group. For b,l, data are presented as mean ± s.d. of n = 3 biological replicates per group. Data in g,i-k are representative of three independent experiments. For i, quantification of the indicated protein level is determined by Image Lab software; data are presented as mean ± s.d. n = 3 independent experiments per group. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (a,b,l), two-way ANOVA with Šídák’s test (d) and two-way ANOVA with Tukey’s test (c,i); ns, not significant.
