Figure 6: Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450.

(a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. Input and bound proteins were detected by Western blotting. (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl₃ and CORM3. (d) Schematic illustration of doxorubicin metabolism is shown on the left. Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. Data represent mean±s.d. of four separate experiments. **P<0.01 versus control using unpaired Student’s t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. Data represent mean±s.d. of four separate experiments. *P<0.01 using ANOVA with Fischer’s LSD test.