Fig 2: SELENOF levels and cellular location in human cell lines. PC3 and LNCaP cells were fixed, permeabilized and immunostained with SELENOF antibodies. SELENOF was visualized with Alexa Fluor 647. ER and nuclei were visualized with ER Tracker Green and DAPI, respectively

Fig 3: SELENOF levels are lower in prostate tumors compared to benign glands and non‐tumorigenic cells in culture. (A) A representative image of a tissue core composed of both tumor and benign glands immunostained with anti‐SELENOF antibodies. Nuclei were counterstained with hematoxylin. (B) Primary prostate cells and RWPE‐1 cells were fixed, permeabilized, and co‐immunostained with SELENOF and E‐Cadherin antibodies. SELENOF was visualized with Alexa Fluor 488 (primary prostate cells) or with Alexa Fluor 647 (RWPE‐1). E‐Cadherin was visualized with Alexa Fluor 647 (primary prostate cells) or with Alexa Fluor 588 (RWPE‐1). Nuclei were visualized with DAPI. Table 1 Distributions of the Selenoprotein Genotypes Overall and by Race/Ethnicity

Fig 4: Loss of SELENOF renders MCF-10A cells resistant to apoptosis and autophagy. (A) Fluorescence images of 3D acini at 4× generated from MCF-10A WT or SELENOF KO cells. All cells were seeded in 5% matrigel for 5 days prior to treatment with −/+ 10 µM ZVAD and −/+ 5 µM CQ, then grown for another 15 days. The first row shows representative images of Hoechst 33,342 staining of nuclei in blue. The second row shows representative images of Calcein AM staining of live cells in green. The third row shows representative images of propidium iodide (PI) staining of dead cell in red. The last row shows a merged image of all three stainings. (B) Cell death was estimated using FACS of cells stained with PI after 24 or 48 h of serum deprivation. ** p < 0.01, # p < 0.0001.

Fig 5: SELENOF overexpression induces apoptosis and autophagy in breast cancer cells. (A) Transmission electron microscopy analysis of MCF-7 SELENOF cell treated with -/+ 1 µg/mL Dox for 72 h. Examples of the observed mitochondrial morphology (top row) and phagosome formation (bottom row) are indicated by arrows. Serum starvation (last column) for 24 h was used as a positive control for autophagy. (B) DNA fragmentation was estimated using Hoechst 33,342 staining in cells treated as described in (A). (C) The levels of SELENOF and LC3-II were determined using Western blotting in whole cell extracts from MCF-7 SELENOF cells treated with 1 µg/mL Dox at the indicated times. Serum starvation for 24 h was used as a positive control for autophagy. ß-Actin is shown as a loading control. The uncropped blots are shown in File S1. (D) Cell viability was determined using crystal violet staining of cells treated with -/+ 1 µg/mL Dox, -/+10 µM ZVAD, and -/+5 µM chloroquine (CQ) for 5 days. Data are normalized to vehicle control and shown as 100%. ** p < 0.01, **** p < 0.0001.