Fig. 1

MIA PaCa-2 pancreatic cancer cells morphology is affected by PGRMC1 phosphorylation status. a PGRMC1-HA proteins constructed for this figure. TMH: Trans-membrane helix. HA: the C-terminal 3x hemaglutinin tag. b Detection of exogenous PGRMC1 expression levels by western blot (upper panel). Equal loading is controlled by quantifying beta actin (lower panel). The results show three totally independent stably transfected cell lines per plasmid from (A). Open arrow: Exogenous PGRMC1-HA (Ex.). Shaded arrow: endogenous PGRMC1 (End.). Filled arrow: beta actin. The molecular weight ladder is Bio-Rad 1610377 Dual Xtra Standards. c Box plots quantification of replicate gels of (B) with signals normalized to beta actin from the same respective lanes. n = 4 lanes for MP and n = 6 for WT, DM and TM (replicates of respective lines 1–3 per condition). There were no significant differences (ns) except for the exogenous band in MP (ANOVA, post-hoc Dunnet’s T3). d Western blot quantification of HA-tagged exogenous PGRMC1, following B but detecting PGRMC1 with anti-HA antibody. The molecular weight ladder is Abcam ab116028 Prestained Protein Ladder. e PGRMC1 mutant protein expression alters MIA PaCa-2 cell morphology. PGRMC1-HA-expressing stable cells (respective lines 1 from B) or MP cells were stained with a FITC-tagged anti-HA antibody (Anti-HA) and imaged by confocal microscopy. DNA was stained with DAPI. Cells were also imaged in differential interference contrast (DIC) microscopy mode. The respective left panels show merged images of all 3 channels. f The rounded phenotype of double and triple mutant (E) was reversed to elongated phenotype after 125 μM ROCKI addition, but not by addition of DMSO vehicle control