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Figure 1

From: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

Figure 1

ORM induces autophagic flux in ovarian cancer cells PA-1 and OVCAR-3. (A) Dose-response curve of ORM in ovarian cancer cell lines PA-1 and OVCAR-3. (B) PA-1 cells were stained with MDC, a marker of acidic cellular compartments after treatment with indicated time periods with IC50 dose of ORM. (C) quantification of MDC-positive dots from (B) as described in Methods. (D) Confocal microscopy images of PA-1 and OVCAR-3 cells treated with IC50 dose of ORM and immunostained with LC3 (an autophagosome marker). (E) PA-1 cells were pre-treated with or without 3-MA (class III PI3K inhibitor; 2.5 mM) or CQ (lysosomal fusion inhibitor; 10 μM), immunostained with LC3 and imaged in confocal microscope. (F) Quantification of LC3 puncta from data such as (E) as described in Methods. (G) PA-1 and OVCAR-3 cells were treated with ORM (IC50 dose) for indicated time-points and probed for autophagy markers LC3 and Beclin 1, quantified in (H). (I) PA-1 and OVCAR-3 cells were pre-treated or not with lysosomal fusion inhibitor BafA1 (100 nM; 2 h) to inhibit fusion of autophagosomes with lysosomes. LC3-II conversion was observed with immunoblotting and quantified. (J) PA-1 cells were transfected with tfLC3 (as described in Methods), treated with ORM for 24 h and imaged in confocal microscope. Scale bars = 20 μm (B); 10 μm (D).

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