Figure 4. Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling.

(a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. Input and bound proteins were detected by Western blotting. (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl₃ and CORM3. (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. (d) HCT116 cells were treated with or without 250 μmol l⁻¹ of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantified by reverse-phase HPLC. The data represent mean±s.d. of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells. (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. (g,h) HCT116 cells were treated with or without EGF, SA, RuCl₃ and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting.