Fig 2: Cellular proliferation is impaired upon over-expression of ALADIN, PGRMC1 or PGRMC2. Equal cell numbers of a wild-type NCI-H295R1-TR, scrambled shRNA and AAAS shRNA (AAAS knock-down) expressing NCI-H295R1-TR cells and of b wild-type NCI-H295R, GFP, GFP-ALADIN, PGRMC2-GFP and GFP-PGRMC1 expressing NCI-H295R cells were seeded 48 h before confluence measurement using live cell imaging on IncuCyte Zoom (Essen BioScience). In case of transient transfection with PGRMC2-GFP and GFP-PGRMC1 the proliferation of fluorescent cells which starts at cell density of about zero was measured. Growth curve analysis and growth constant k (slope of regression curve) calculation was done using multilevel regression technique using R Studio [58, 59]

Fig 3: PGRMC2 interacts with ALADIN determined by IP-western and reciprocal IP-western assays. (A) Whole cell lysates of GFP-ALADIN, PGRMC2-GFP- and GFP- (as negative control) expressing NCI-H295R cells were used and GFP pulldown performed followed by western blot with indicated antibodies. PGRMC2 (24 kDa, arrow) could be detected after GFP-ALADIN (86 kDa) pulldown. ALADIN (59 kDa, arrow) and PGRMC2 could be both detected after PGRMC2-GFP (51 kDa) pulldown. GFP (27 kDa) was ascertained after GFP control pulldown but the control remained empty for PGRMC2 and ALADIN. (B) Whole cell lysates of NCI-H295R cells were used for ALADIN and PGRMC2 pulldown. Normal mouse (m IgG) and rabbit IgGs (Rb IgG) served as negative control. Western blot was performed with indicated antibodies. Non-bound (NB) IP fractions are also shown for each pulldown and bound IP eluates are separated by one lane each from the specific controls to eliminate false positive detection. PGRMC2 (arrow) precipitated in endogenous ALADIN pulldown. Control m IgG pulldown remained empty. ALADIN (arrow) reciprocally precipitated after PGRMC2 pulldown. Control Rb IgG pulldown remained empty showing a cross-reactive band a few kDa higher than ALADIN.

Fig 4: Depletion of ALADIN affects PGRMC2 localisation at the nuclear envelope/perinuclear ER in human skin fibroblasts. NCI-H295R1-TR/AAAS knock-down, NCI-H295R1-TR/Scrambled shRNA, NCI-H295R1-TR cells and skin fibroblasts of a triple A patient (homozygous mutation in Exon 9, c.884G>A, p.Trp295X) and of an anonymised control were stained with mouse anti-ALADIN (green), mouse anti-PGRMC2 (green) and DAPI (blue). Scale bars for anti-ALADIN staining (upper panels): 38 µm (NCI-H295R1-TR/AAAS knock-down), 24 µm (NCI-H295R1-TR/Scrambled shRNA), 32 µm (NCI-H295R1-TR cells) and 38 µm (skin fibroblasts). All scale bars for anti-PGRMC2 staining: 24 µm. NCI-H295R1-TR cells with AAAS knock-down or scrambled shRNA were induced as described previously (Jühlen et al., 2015).

Fig 5: PGRMC2 was identified in mass spectrometry after GFP-(ALADIN) pulldown. Identified exclusive unique peptides (yellow) (number of different amino acid sequences, regardless of any modification, that are associated only with this protein) of ALADIN and progesterone receptor membrane component 2 (PGRMC2) are highlighted. Also for PGRMC2, relative intensities of annotated spectra are shown after GFP(-ALADIN) co-IP of whole cell lysates of GFP-ALADIN expressing NCI-H295R cells.