Fig 2: PERP protein is stabilized by ER stress, independent of p53 transcriptional regulation.HCT116 cells were treated with 1 μg/ml BFA for the indicated time points and changes to the levels of a SERCA2b mRNA (n = 4, one-way ANOVA, F = 3.442, p = 0.0239*), b SERCA2b protein (n = 7), c PERP protein (n = 7, one-way ANOVA, F = 3.025, p = 0.0329*) and d PERP mRNA (n = 4, one-way ANOVA, F = 5.297, p = 0.0036**) were detected by RT-PCR or Western blot and normalized to the level of GAPDH. e HCT116 p53−/− cells were treated with 1 µg/ml BFA and the response of PERP protein levels was detected by immunoblotting

Fig 3: PERP interacts with SERCA2b at ER–PM junctions.a Protein interacting partners of Halo-PERP were isolated from Mel202 cells using the HaloTag Mammalian Pull-Down System and identified by mass spectrometry. Maxquant intensity values for SERCA2 and PERP in three independent experiments are shown. b The interaction of PERP and SERCA2b was confirmed in two independent HaloTag pull-down experiments by immunoblotting as described in the Methods (proteins eluted from the resin by TEV enzymatic cleavage followed by a successive SDS-based elution). Figure shows two different exposures of SERCA2b panel to allow visualization of lower intensity bands. c PERP–SERCA2 complex formation was validated by SERCA2 IP in Mel202 cells expressing HaloTag or Halo-PERP using a HaloTag antibody. d Diagram of SERCA2a–c proteins, indicating the relative positions of the peptide identified by mass spectrometry (black) and the antibody immunogen sites (grey) used for validation of the SERCA2b–PERP interaction. Antibody 1 was used in HaloTag pull down validation (b) and antibody 2 was used for IP of SERCA2 (c). e Super-resolution images of HeLa BAC Venus-PERP cells co-expressing mCherry-SERCA2b, junctions between the ER and PM shown by arrows. Scale bar: 20 µm in full image and 5 µm in zoom panel

Fig 4: Trastuzumab upregulates PERP in extracellular vesicles emitted by trastuzumab-sensitive but not by trastuzumab-resistant human breast tumor cells. BT-474 (a) or BT-474TR (b) cells were treated (+) or not (−) with 5 μg/ml trastuzumab (TZ) in 3D culture for 72 h. Extracellular vesicles were isolated from the cell media by ultracentrifugation and assayed for PERP levels by western blot. An extracellular vesicle marker Flotillin-1 is a loading control. Trastuzumab-sensitive MCF7/Her2-18 (c) and trastuzumab-resistant HCC-1419 (d) human ErbB2-positive breast cancer cells were treated (+) or not (−) with trastuzumab for 48 h (c) or 72 h (d), and extracellular vesicles were isolated from the cell media as in a and analyzed as in a. Bar graphs to the right of the western blot images represent quantification of respective bands. PERP levels were normalized by the levels of the loading control. PERP levels (relative level) in the control cells were designated as 1.0. The data represent the average of five (a), three (b, c), and two (d) independent experiments, plus SE. *p value was < 0.05

Fig 5: PERP and/or GNAS2 and/or GNA13 and/or ITB1 and/or RAB10 are upregulated in the blood extracellular vesicles of patients with ErbB2-positive metastatic breast cancer that benefited from trastuzumab-based therapies but not in those derived from patients that failed these treatments. Blood was collected from the indicated patients on the indicated days during trastuzumab (TZ)-based therapies. The day of the first blood collection was designated as “day 0.” Normally, blood was collected 1 day prior to the therapy. The extracellular vesicles were isolated from patients’ blood by ultracentrifugation and tested for the levels of the indicated proteins by western blotting. Extracellular vesicle markers Flotillin-1 and TSG101 are loading controls. Patients’ metastatic disease status (shown above each blot) was assessed based on the comparison of their CT scans performed as closely as possible to the dates of the first and the last blood collection. Data for patients showing partial response or stable disease (a) or those with progressive disease (b) are shown