Fig 2: EWI-2 localizes on the nuclear envelope. (A) PC3 and Du145 cells were labeled with Alexa 488-conjugated lamin A/C mAb and EWI-2 pAb, then Alexa 647-conjugated secondary antibody. Images were captured with confocal fluorescence microscopy. Scale bars: 10 µm. Insets: nuclear colocalization of EWI-2 with Lamin A/C in PC3 and Du145 cells. Scale bars: 1 µm. N, NEG; K, KO. (B) Total and NE proteins were extracted from the PC3 and DU145 cells and examined for EWI-2 and other indicated proteins in western blot assay. Lamin A/C was used as the marker of NE and EEA1 served as the marker of cytoplasm. (C) The band densities of (B) were quantified using Student’s t-test and are presented as mean ± SD (n = 3 individual experiments). *P < 0.05; ***P < 0.001. (D) Immunohistochemistry analysis on EWI-2 proteins in human normal prostate and prostate cancer tissues. The tissues were co-stained with hematoxylin for nuclei. Scale bar: 50 µm. (E,F) Expression of EWI-2/IGSF8 in prostate adenocarcinoma based on metastasis status (D) and Gleason score (E) in TCGA database (http://ualcan.path.uab.edu/cgi-bin/ualcan-res.pl). The expression level of EWI-2/IgSF8 between groups was compared via Student’s t-test for multiple groups and the error bar was presented with the confidence interval (CI). The precise P-values of (D,E) are presented in Supporting Information Fig. S4. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: EWI-2 expression in extracellular vesicles. (A) Western blot analysis of EWI-2 protein levels in PC3 cells upon EWI-2 knockout. Actin serves as a protein loading control. The normalized band densities were quantified with Student’s t-test and are presented as mean ± SD (n = 3 individual experiments). **P < 0.01. (B) EWI-2-CRISPR/Cas9 knockout efficiency in PC3 cells, as analyzed with flow cytometry. Student’s t-test was used for statistical analysis on fluorescence intensity (MFI) (mean ± SD, n = 3 individual measurements). **P < 0.01. (C) Extracellular staining of EWI-2 in PC3 cells. After the cells were fixed, permeabilized and stained for EWI-2 mAb, F-actin with phalloidin, and nucleus with DAPI, immunofluorescence images were captured with confocal fluorescence microscopy. Scale bar: 10 µm. (D) Cell surface CD9, CD81 and integrin aV expression in PC3 cells, as analyzed with flow cytometry. Student’s t-test was used for statistical analysis on MFI (mean ± SD, n = 3 individual measurements; *P < 0.05). (E) The exosomes were extracted and examined with western blot analysis. The membrane were probed with EWI-2 and exosome marker antibody (anti-CD54, anti-CD9, anti-flotillin, anti-EpCam and anti-annexin antibody). (F) The normalized band densities of PC3-EWI-2-CRISPR/Cas9 knockout cells and DU145-EWI-2-CRISPR/Cas9 knockout cells in (E) were quantified with Student’s t-test and presented as mean ± SD (n = 3 individual experiments). *P < 0.05; **P < 0.01; ***P < 0.001. (G) The normalized band densities of PC3-EWI-2-overexpression cells were quantified with Student’s t-test and presented as mean ± SD (n = 3 individual experiments). *P < 0.05. (H) Western blot analysis of cellular expression of Caveolin-1, PTRF (Cavin-1) and Flotillin-1, together with actin as a loading control of whole-cell lysates. The band density of KO cells in each individual experiment was normalized with the density of NEG cells. The normalized band densities were quantified with Student’s t-test and presented as mean ± SD (n = 3 individual experiments). *P < 0.05; ***P < 0.001.

Fig 4: EWI-2 removal potentiates EGFR-MAPK signaling. (A) The EGFR activity was analyzed with Western blot in PC3 cells, together with actin as a loading control of whole-cell lysates. (B) The normalized band densities were quantified with Student’s t-test and presented as mean ± SD (n = 3 individual experiments). **P < 0.01. (C) EWI-2 knockout resulted in higher EGFR clustering at the cell surface in PC3 cells. The cells were treated with EGF (25 nm) for 2 min, fixed, and stained with Alexa 647-conjugated EGFR antibody. The EGFR distribution on the cell surface was imaged at nano-scale by N-STORM super-resolution microscopy. Scale bars: 1 µm. (D) Ripley’s H functions were acquired by nis-elements AR software (left), and the r max of H(r) between groups was compared via independent samples t-test (right) (mean ± SD, n = 3 independent experiments). ***P < 0.001. (E) EGF-based endocytosis of EGFR was analyzed with 2 µg·mL–1 pHrodo green EGF conjugate in LCIS with incubation for 15–20 min at 37 °C. Cells were then washed in LCIS and fixed. Cells were imaged under N-STORM imaging modality (Nikon) confocal microscope. (F) The mean fluorescence intensity of internalized green EGF was analyzed with imagej software and compared between groups statistically using Student’s t-test (mean ± SD, n = 3 independent experiments). *P < 0.05. (G) Antibody-based endocytosis of EGFR. The cells were incubated with EGFR mAb (1 µg·mL–1) and then incubated at 37 °C for 15–20 min, followed by three washes of 0.1 m glycine/1.0 m NaCl solution (pH 2.5). The cells were then fixed, permeabilized, incubated with secondary antibody and imaged with confocal microscopy. Scale bar: 10 µm. (H) The mean fluorescence intensity of internalized EGFR was analyzed with imagej software and compared between groups statistically using Student’s t-test (mean ± SD, n = 3 independent experiments). ***P < 0.001. (I) The cells were fixed, permeabilized, incubated with ERK1/2 antibody and then fluorochrome-conjugated 2nd antibody, and imaged with confocal microscopy. Scale bar: 10 µm. (J) The percentage of cells with nuclear ERK1/2 staining was quantified using Student’s t-test and presented as mean ± SD (n = 3 individual experiments). ***P < 0.001.

Fig 5: The role of miR-7 in Sim1 neurons to regulate mammalian energy homeostasis and neuroendocrine function.In mice, hypothalamic expression of miR-7 is regulated by metabolic state. In humans, variants in the locus encompassing miR-7 are associated with reduced expression of HNRNPK/MIR-7-1. miR-7 regulates the expression of the noncoding RNAs Cyrano and Cdr1as (dashed lines indicate previously published data35). Loss of miR-7 expression in Sim1 neurons leads to upregulation of miR-7 target genes, including Snca and Igsf8, to disrupt the function of multiple Sim1 neuron subpopulations. As a result, the absence of miR-7 causes hyperphagic obesity, increased linear growth, increased insulin secretion, increased fluid intake, and suppressed HPA axis function. Green arrows indicate a positive effect on expression; red blunt-headed lines indicate a negative effect on expression.