Fig 2: Expression of gelsolin is associated with poor clinical outcomes in patients with PCa(A) Representative examples of gelsolin (red crimson precipitates) expression in prostate cancer (PCa) cells, which was detected by immunohistochemistry, and the slide was counterstained with hematoxylin. In non-tumor prostate epithelial cells, secreted gelsolin was detected in the lumens (original magnification × 100). (B) Compared to hypertrophic region, expression of gelsolin was detected in tumor nests (Gleason score 6) (original magnification × 100). (C) In pathological tissue of PCa specimens with higher Gleason score, overexpression of gelsolin was detected in all the PCa cells (Gleason score 9) (original magnification × 100). (D) In PCa tissue with metastatic lesions, overexpression of gelsolin was detected in tumor nest and the metastatic PCa cells (Gleason score 9) (original magnification × 100). (E) In bone marrow section of PCa specimens, gelsolin was mainly detected in the invasive PCa cells (original magnification × 100). (F) Around tumor nest, in which gelsolin was minimally expressed, a swarm of lymphocytes without evident gelsolin signal were surrounding the tumor nest (original magnification × 100). (G) In some areas, many cells with darkly stained nuclei in the tumor nest were positive for gelsolin. However, nuclear morphologies of these cells were different from the neighboring PCa cells (original magnification × 400). (H) Similar to cells, which had darkly stained nuclei in PCa tumor nest, the cells were CD3-positive (original magnification × 400). (I) Expression of gelsolin in PCa and benign hypertrophic prostate epithelia (BHPE) cells was confirmed by Western blotting analysis. (J) Kaplan-Meier plots of overall survival in PCa patients with high (GSNHigh) or low gelsolin (GSNLow) expression. (K) Survival analysis of PCa patients in whom lymphocytes were positive (GSN+LYM+) or negative (GSN-LYM-) for gelsolin staining. Detection of gelsolin staining correlated with poor prognosis.

Fig 3: Validation of GSN and LDHA. (A) Relative protein expression levels of GSN and LDHA from iTRAQ data. Results are expressed as fold change, and protein expression levels were normalized to 2D culture. Values are shown as the means ± SEM. GSN, gelsolin; LDHA, lactate dehydrogenase A. (B) Validation of GSN and LDHA in 2D- and 3D-cultured SW480 cells either treated or untreated with XAV939 using western blot analysis. Full-length blots are presented in Supplementary Fig. S4. (C) Representative MS/MS spectra of AQPVQVAEGSEPDGFWEALGGK and QTQVSVLPEGGETPLFK for identified GSN and DLADELALVDVIEDK and DQLIYNLLK for identified LDHA. Left boxes represent spectra of iTRAQ reporter ions. 113.11, 2D culture; 114.11, 2D culture treated with XAV939; 115.11, 3D culture; 116.11, 3D culture treated with XAV939.

Fig 4: ABT-737-induced caspase-3 cleavage in mouse platelets is unchanged by the loss of gelsolin.A. Washed wild-type (WT) and gelsolin-null (Gsn-/-) platelets were treated with DMSO vehicle alone, with 1 μM ABT-737 or with 10 μM ABT, for 60 or 120 minutes as indicated. Equal amounts of lysate were resolved by SDS-PAGE; immunoblots were probed for pro-caspase-3 and cleaved caspase-3. Beta-actin (β-actin) and beta-tubulin (β-tubulin) are shown as loading controls. B. Bar graph depicts caspase-3 cleavage (normalized to beta-tubulin levels) in wild-type (WT, grey bars) and gelsolin-null (Gsn-/-, black bars) platelets were treated with DMSO vehicle alone, with 1 μM ABT-737 or with 10 μM ABT, and for 60 or 120 minutes as indicated. Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments.

Fig 5: ABT-737-induced mitochondrial depolarization and PS externalization in mouse platelets is unchanged by the loss of gelsolin.A. Western blot confirms the deletion of gelsolin expression in mouse platelets (WT, wild-type; Gsn-/-, knockout). β-tubulin is shown as a loading control. B. Mitochondrial depolarization was measured in washed mouse wild-type (WT, grey bars) and gelsolin-null (Gsn-/-, black bars) platelets using the JC-1 dye. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone, with 1 μM ABT-737 or with 10 μM ABT, for 60 or 120 minutes as indicated. Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments. C. Phosphatidylserine (PS) exposure was measured in washed mouse wild-type (WT, grey bars) and gelsolin-null (Gsn-/-, black bars) platelets using the Annexin V dye. Bar graphs depict PS exposure in platelets treated with DMSO vehicle alone, with 1 μM ABT-737 or with 10 μM ABT, for 60 or 120 minutes as indicated. Data are mean ± SEM, analyzed by using 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments.