Journal: Frontiers in Immunology

Article Title: Identify GDPD3 as a key regulator of epithelial–mesenchymal transition and prostate adenocarcinoma progression via the LPA/LPAR1/AKT axis: transcriptomic and experimental study

doi: 10.3389/fimmu.2025.1637325

Figure Lengend Snippet: GDPD3 promotes EMT through LPA–LPAR1 signaling. (A) Analysis of the correlation between GDPD3 and LPA. (B) LPA levels were measured by ELISA following GDPD3 overexpression in DU145 cells. (C) DU145 cells were treated with either vehicle control or 20 μM LPA for 24, 48, and 72 hours. Protein levels of N-cadherin, E-cadherin and Vimentin were assessed by Western blot and quantified. (D, E) DU145 cells were treated with LPA alone, or with LPA in combination with KI6425 or AM095 (both LPAR1 inhibitors). EMT markers N-cadherin, Vimentin and α-SMA were evaluated by Western blot, and band intensities were quantified using ImageJ software, normalized to actin. (n = 3–4 independent experiments). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The DU145 human prostate cancer cell line was obtained from the American Type Culture Collection (ATCC) in February 2025.

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Control, Western Blot, Software

Journal: Frontiers in Immunology

Article Title: Identify GDPD3 as a key regulator of epithelial–mesenchymal transition and prostate adenocarcinoma progression via the LPA/LPAR1/AKT axis: transcriptomic and experimental study

doi: 10.3389/fimmu.2025.1637325

Figure Lengend Snippet: LPAR1 knockdown and AKT inhibition attenuate LPA-induced EMT. (A, B) LPAR1 was knocked down, and knockdown efficiency was subsequently validated. (C, D) DU145 cells were transfected with siRNA targeting LPAR1, and the expression levels of N-cadherin, E-cadherin and Vimentin were assessed by Western blot. (E, F) Western blot analysis was performed to assess the expression levels of AKT and phosphorylated AKT (p-AKT). (G, H) DU145 cells were treated with 2 μM LY294002 (AKT pathway inhibitor), and the protein levels of AKT, phosphorylated AKT (p-AKT), N-cadherin, E-cadherin and Vimentin were assessed by Western blot and quantified. Band intensities were quantified using ImageJ software and normalized to actin. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The DU145 human prostate cancer cell line was obtained from the American Type Culture Collection (ATCC) in February 2025.

Techniques: Knockdown, Inhibition, Transfection, Expressing, Western Blot, Software

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: Characterization of PCa EVs. (A) EV size distribution measured by nanoparticle tracking analysis. (B) EV morphology visualized by transmission electron microscopy. Scale bars are 300 nm. (C) Western blot analysis was performed to investigate the expression levels of TSG101, Alix, Hsc70, CD9, calnexin, and cytochrome c in PC3 and DU145 cells and in their EVs. One representative of three experiments performed is shown.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: PCa EVs promote lipolysis in adipocytes. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. Lipid accumulation was then evaluated by cytofluorimetric analysis after staining with Bodipy 1 μM for 30 min. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. FFA release was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (C) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. Glycerol release was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (D) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 6 h. cAMP levels were then evaluated by ELISA assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (E) After PCa EV treatment (30 μg/mL for 12 h), Western blot analysis was performed to investigate the expression levels of PKA substrates, HSL, G0S2, and ATGL in 3T3‐L1 adipocytes. GAPDH expression was evaluated as a loading control. One representative of three experiments performed is shown. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Staining, Control, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: The secretome from EV‐treated adipocytes increases PCa cell growth. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (96 h). Cell proliferation was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control). (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (72 h). Clonogenic ability was then evaluated by colony formation assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control), ** p < 0.01 vs. Adipo CM (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Trypan Blue Exclusion Assay, Control, Colony Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: The secretome from EV‐exposed adipocytes enhances PCa cell invasive potential. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by wound healing assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05. Scale bars are 200 μm. (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control), *** p < 0.001 vs. Adipo CM (control). (C) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (48 h). Anoikis was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control). (D) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell invasion was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. Adipo CM (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Migration, Wound Healing Assay, Transwell Assay, Control, Trypan Blue Exclusion Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: FFAs in EV‐conditioned adipocyte secretome are taken up by PCa cells and mediate the above pro‐tumor effects. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). FFA uptake was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Dunnet's test. *** p < 0.001. (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Lipid accumulation was then evaluated by cytofluorimetric analysis after staining with Bodipy 1 μM for 30 min. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. *** p < 0.001 vs. C (control). (C) After delipidation, EV‐Adipo CM was given to PC3 and DU145 cells (72 h). Cell proliferation was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, ** p < 0.01. (D) After delipidation, EV‐Adipo CM was given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Colorimetric Assay, Staining, Control, Trypan Blue Exclusion Assay, Migration, Transwell Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: FFAs in EV‐conditioned adipocyte secretome trigger the Akt signaling in PCa cells. (A) After EV‐Adipo CM treatment (24 h), Western blot analysis was performed to investigate the expression levels of p‐Akt in PC3 and DU145 cells. GAPDH expression was evaluated as a loading control. One representative of three experiments performed is shown. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. *** p < 0.001 vs. C (control). (B) After BEZ235 treatment (100 nM, 72 h) following EV‐Adipo CM treatment (3 h), PC3 and DU145 cell proliferation was evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, *** p < 0.001. (C) After BEZ235 treatment (100 nM, 24 h) following EV‐Adipo CM treatment (3 h), PC3 and DU145 cell migration was evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, *** p < 0.001.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Western Blot, Expressing, Control, Trypan Blue Exclusion Assay, Migration, Transwell Assay