Fig 2: TSPO is colocalized with SW120 receptor S2R in MP cells. The left panels represent PLA results for TMEM97 (TMEM) and TSPO pairing. The positive signal was observed compared with the negative signal after attenuating TSPO. The right panels show two negative PLA signals for TMEM97 + PGRMC1 and PGRMC1 + TSPO. Those negative observations were similar to a negative control without primary antibodies. Red corresponds to the signal amplified by the Duolink PLA reaction described in Section 4.12. Blue represents DAPI staining of DNA. Each discrete blue patch represents one cell nucleus (imaged at 20× magnification).

Fig 3: 20S-OHC reduces levels of both TMEM97 and PGRMC1 co-immunoprecipitated with TSPO. MP cells were treated for 24 h with 20S-OHC, immunoprecipitated with an antibody directed against TSPO, and the immune pellets were analyzed by Western blot with anti-TMEM97 (A) or anti-PGRMC1 (B) specific antibodies. IgG is the negative control for the anti-TSPO experimental treatment immunoprecipitation. Western blotting data are representative of at least three independent experiments. TMEM97 (A) and PGRMC1 (B) protein levels in 20S-OHC treated cells were normalized versus proteins in untreated cells (C) and reported in the respective graph bars as means ± SD (error bars). Statistical significance of the differences was evaluated by using Student’s t-test (** p < 0.01, *** p < 0.001).

Fig 4: Confocal microscopy on HeLa cell lines labeled with antibodies against LDLR, PGRMC1, and TMEM97. (A) Cells were plated and imaged under standard, non-lipoprotein depleted conditions. (B) Cells were plated and sterol starved by incubation with lipoprotein-depleted serum for 24 hours. (C) Cells were sterol starved for 24 hours, followed by treatment with 50 µg/mL LDL for 3 hours.

Fig 5: TSPO co-immunoprecipitates only with TMEM97 in MCF7 cells and with both TMEM97 and PGRMC1 in MP cells. MCF7 and shTMEM97MCF7 cells were immunoprecipitated with an antibody directed against TSPO and analyzed by Western blot with anti-TMEM97 (A) or anti-PGRMC1 (B) antibodies. TMEM97 protein levels in shTMEM97MCF7 cells were normalized versus proteins in MCF7 cells. MP cells were immunoprecipitated with an antibody directed to TSPO and analyzed by Western blot with anti-TMEM97 (C) or anti-PGRMC1 (D) antibodies. IgG is the negative control. Western blot analyses were performed to evaluate the protein levels of TMEM97 (E) and PGRMC1 (F) in MCF7 MP cells. In (E,F) protein levels were normalized against ß-actin. Western blotting data are representative of at least three independent experiments. Graph bars were used to represent the means ±SD (error bars). Statistical significance of the differences was evaluated by using Student’s t-test (* p < 0.05, *** p < 0.001).