Journal: Molecular Endocrinology

Article Title: Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

doi: 10.1210/me.2012-1079

Figure Lengend Snippet: The co-culture with macrophages can increase prostate epithelial cell growth in a 3D culture model. BPH-1 cells were cultured in a 3D culture condition with the co-cultured medium of BPH-1/THP-1 cells or control medium. A (magnification, ×10), The co-cultured medium of BPH-1/THP-1 cells increased the sphere diameter of BPH-1 cells (scale bar, 20 μm), as shown in gross observation of BPH-1 spheres under mono-culture (upper) and co-culture (lower). B, Results are expressed as the average size of the spheres (sphere number: 30 for each experiment set) for three independent experiments. The diameter difference of spheres was analyzed by two-tailed Student's t test. C, (magnification, ×100), mPrE cells were cultured in a 3D culture condition, and the results of the sphere diameter of mPrE cells were expressed as described in panel B; scale bar, 100 μm.

Article Snippet: Cell culture and co-culture experiments Human benign prostate epithelial cell line, BPH-1, was purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Co-Culture Assay, Cell Culture, Control, Two Tailed Test

Journal: Molecular Endocrinology

Article Title: Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

doi: 10.1210/me.2012-1079

Figure Lengend Snippet: The coculture with macrophages induces EMT gene expression in prostate epithelial cells in 3D culture condition. A, Quantitative PCR analysis of EMT markers in BPH-1 cells. N-cadherin, Snail, and TGF-β2 were increased after co-culture. E-Cadherin expression was decreased. Bars represent an average expression level (fold) of three independent experiments. Error bars represent sem. *, P < 0.01. The difference of gene expression was analyzed by two-tailed Student's t test. B, Western blot analysis of EMT markers in BPH-1 cells. C, Western blot analysis of EMT markers, Snail and N-cadherin, in mPrE cells with or without coculture with RAW264.7 macrophages.

Article Snippet: Cell culture and co-culture experiments Human benign prostate epithelial cell line, BPH-1, was purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Co-Culture Assay, Expressing, Two Tailed Test, Western Blot

Journal: Molecular Endocrinology

Article Title: Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

doi: 10.1210/me.2012-1079

Figure Lengend Snippet: The expression of AR in prostate epithelial cells increased THP-1 cell migration, sphere number, and EMT induction. A, The expression of AR in BPH cells enhanced the migration of THP-1 cells during co-culture. The THP-1 migration assay was performed as described in Fig. 2. Briefly, THP-1 cells were co-cultured with BPH-V or BPH-AR cells. Bars represent an average number of migrated cells of three independent experiments (left panel; **, P = 0.003). The sphere formation assay was performed as described in Fig. 3 to examine the 3D growth of BPH-V and BPH-AR cells (middle panel; *, P = 0.0022). Results are shown as described in Fig. 3. AR expression was confirmed by Western blot analysis (right panel). B, Quantitative PCR and Western blot analysis of Snail, N-cadherin, and E-cadherin expression in BPH-v and BPH-AR cells (left and middle panels); *, P < 0.01. Western blot analysis of EMT markers in mPrE-v and mPrE-AR cells after coculture (right panel). C, ASC-J9 can suppress AR expression in BPH-AR cells and inhibit BPH-AR sphere formation in the presence of coculture medium. P values are shown as indicated. D, ASC-J9 can block induction of EMT markers in BPH-AR cells. DMSO, Dimethylsulfoxide.

Article Snippet: Cell culture and co-culture experiments Human benign prostate epithelial cell line, BPH-1, was purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Migration, Co-Culture Assay, Cell Culture, Tube Formation Assay, Western Blot, Real-time Polymerase Chain Reaction, Blocking Assay

Journal: Frontiers in immunology

Article Title: Reducing farnesyl diphosphate synthase levels activates Vγ9Vδ2 T cells and improves tumor suppression in murine xenograft cancer models.

doi: 10.3389/fimmu.2022.1012051

Figure Lengend Snippet: FIGURE 1 A lentivirus expressing a short-hairpin RNA (shRNA) targeting farnesyl diphosphate synthase (FDPS) reduces its expression in cancer cells. (A) Schematic diagram of the lentivirus vectors LV-shFDPS and LV-shFDPS-IL2. Additionally, the vectors may express the luciferase and GFP gene. (B, C) Real-time qPCR analysis of FDPS RNA from PC3 prostate and Huh-7 hepatocellular carcinoma cells transduced with LV-shFDPS at a MOI of 5, 10, and 20 or 25. In PC3 cells at a MOI of 25, FDPS expression was reduced by 74% and in Huh-7 cells at a MOI of 20, FDPS expression was reduced by 84%. (D, E) Immunoblot analysis of FDPS protein from PC3 and Huh-7 cells transduced with LV-shFDPS.

Article Snippet: Cultivation of cancer cell lines PC3 prostate carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and Huh-7 liver hepatocellular carcinoma cells were obtained from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan).

Techniques: Expressing, shRNA, Luciferase, Transduction, Western Blot

Journal: Frontiers in immunology

Article Title: Reducing farnesyl diphosphate synthase levels activates Vγ9Vδ2 T cells and improves tumor suppression in murine xenograft cancer models.

doi: 10.3389/fimmu.2022.1012051

Figure Lengend Snippet: FIGURE 2 Vd2 T cells increase cytokine production and cytotoxic activity when cultured with cancer cells treated with low-dose zoledronic acid (ZA) and transduced with shFDPS. (A) Representative flow cytometry dot plots from PC3 and Huh-7 cells transduced with LV-shFDPS in the presence or absence of ZA for overnight, followed by co-culture with Vd2 T cells for 4 h. The gated region indicates the Vd2 and IFNg positive population. In PC3 cells, the percent positive of IFNg producing cells was 9.59 (LV + ZA), 1.28 (LV-shFDPS), and 30.9 (LV-shFDPS + ZA). In Huh-7 cells, the percent positive of IFNg producing cells was 38.9 (LV + ZA), 49.3 (LV-shFDPS), and 76.3 (LV-shFDPS + ZA). (B, C) LV-shFDPS transduced PC3 and Huh-7 cells were treated either with or without ZA for overnight, followed by co-culture with Vd2 T cells for 4 h. The cells were analyzed by flow cytometry for Vd2 and TNFa expression. The data represents a summary of assays using Vd2 T cells from multiple donors (PC3, N=56; Huh-7, N=39). Each dots represents the percentage of Vd2 and TNFa positive cells from an individual donor. In PC3 cells, the percent positive of TNFa producing cells was 12 ± 10.9% (LV + ZA), 4.8 ± 4.3% (LV-shFDPS), and 38.6 ± 16.3% (LV-shFDPS + ZA). In Huh-7 cells, the percent positive of TNFa producing cells was 16.1 ± 15.9% (LV + ZA), 20 ± 10.8% (LV-shFDPS), and 36.9 ± 23.1% (LV-shFDPS + ZA). (D, E) LV or LV- shFDPS transduced PC3 and Huh-7 were treated either with or without ZA for overnight as the target cells and labeled with calcein AM. This was followed by culturing the target cells with a serial dilution of Vd2 effector cells in target ratios (E/T) for 4 h. All co-cultures were performed in triplicate. Cell lysis was measured by the level of fluorescent calcein AM detected in the supernatant.

Article Snippet: Cultivation of cancer cell lines PC3 prostate carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and Huh-7 liver hepatocellular carcinoma cells were obtained from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan).

Techniques: Activity Assay, Cell Culture, Transduction, Cytometry, Co-Culture Assay, Expressing, Labeling, Serial Dilution, Lysis

Journal: Frontiers in immunology

Article Title: Reducing farnesyl diphosphate synthase levels activates Vγ9Vδ2 T cells and improves tumor suppression in murine xenograft cancer models.

doi: 10.3389/fimmu.2022.1012051

Figure Lengend Snippet: FIGURE 3 Vd2 T cells suppress the growth of PC3 prostate carcinoma tumors transduced with a lentivirus expressing a shRNA targeting FDPS. (A) Vd2 T cells significantly suppressed the growth of PC3 tumors transduced with LV-shFDPS as compared with LV (**p<0.0037, N=8). At the end of the study, 36 days post Vd2 injection, the average tumor volume for LV-shFDPS was 1680 ± 166 mm3 which was reduced to 795 ± 193 mm3 when combined with Vd2 treatment. There was minimal effect on tumor volume with ZA treatment with or without LV-shFDPS. (B) An analysis using a Kaplan-Meier survival curve was based on the end event being when the tumor size reached 2000 mm3. There was a significant survival advantage in mice with PC3 tumors transduced with LV-shFDPS and treated with Vd2 T cells as compared with no Vd2 T cells (****p < 0.0001, N=8). All mice with LV transduced PC3 tumors with or without treatment of Vd2 T cells were sacrificed up to day 22. In mice with LV-shFDPS transduced PC3 tumors, mice survived up to day 44, and with Vd2 T cell treatment all mice survived up to day 60. (C, D) Mice were imaged for luciferase expression with a Xenogen IVIS200 bioluminescent imager. All tumor groups showed a similar photon intensity 11 days after the initial injection of Vd2 T cells. On day 28, in the mice injected four times with Vd2 T cells, the photon intensity of LV-shFDPS transduced PC3 tumors as compared with LV was significantly decreased from 1.4 X 107 to 2.5 X 104 photon units (**p=0.002, N=6). (E) Comparison of LV and LV- shFDPS transduced PC3 tumors in combination with Vd2 T cell treatment. At the end of the study, each group of mice (N=7 or 8) were euthanized, tumors were extracted and weighed. In mice injected four times with Vd2 T cells, the tumor weight of LV-shFDPS transduced PC3 tumors was significantly decreased as compared with LV from 1.5 to 0.5 g (***p=0.0002, N=8). ns, not significant.

Article Snippet: Cultivation of cancer cell lines PC3 prostate carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and Huh-7 liver hepatocellular carcinoma cells were obtained from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan).

Techniques: Transduction, Expressing, shRNA, Injection, Luciferase, Comparison

Journal: Frontiers in immunology

Article Title: Reducing farnesyl diphosphate synthase levels activates Vγ9Vδ2 T cells and improves tumor suppression in murine xenograft cancer models.

doi: 10.3389/fimmu.2022.1012051

Figure Lengend Snippet: FIGURE 4 Vd2 T cells suppress the growth of PC3 prostate carcinoma tumors transduced with a lentivirus expressing IL-2 and a shRNA targeting FDPS. (A) ELISA analysis of secreted IL2 expression by PC3 cells transduced with LV-shFDPS-IL2. (B) Representative flow cytometry dot plots from PC3 cells transduced with LV-shFDPS or LV-shFDPS-IL2, followed by co-culture with Vd2 T cells for 4 h. The gated region indicates the Vd2 and IFNg positive population. The percent positive of IFNg producing cells was 10.7 (LV-shFDPS) and 16.6 (LV-shFDPS-IL2). (C) Tumor volume of PC3 cells transduced with LV-shFDPS or shFDPS-IL2. In mice treated with Vd2 T cells, the tumor volume of PC3 cells transduced with LV-shFDPS was significantly decreased at the end of the study from 1900 mm3 to 356 mm3 and with LV-shFDPS-IL2 from 1835 mm3 to 116 mm3 (****p=0.0001, ***p=0.001, N=8). There was no significant difference when comparing LV-shFDPS and LV-shFDPS-IL2 (ns p=0.743, N=8). (D) Comparison of LV, LV-shFDPS, and LV-shFDPS-IL2 transduced PC3 tumors in combination with Vd2 T cell treatment. At the end of the study, each group of mice (N=8) were euthanized, tumors were extracted and weighed. There was a significant decrease in tumor weight of PC3 tumors transduced with LV- shFDPS when treated with Vd2 T cells from 1.25 g to 0.23 g (5.5-fold decrease, ****p<0.0001, N=8). The addition of IL2 in the LV-shFDPS vector also significantly decreased tumor weight in mice treated with Vd2 T cells as compared with LV-shFDPS-IL2 alone from 1.31 g to 0.04 g. There was also a significant 6-fold decrease in the tumor weight of LV-shFDPS-IL2 vs LV-shFDPS groups with Vd2 T cells (* p=0.012, N=8). ns= not significant.

Article Snippet: Cultivation of cancer cell lines PC3 prostate carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and Huh-7 liver hepatocellular carcinoma cells were obtained from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan).

Techniques: Transduction, Expressing, shRNA, Enzyme-linked Immunosorbent Assay, Cytometry, Co-Culture Assay, Comparison, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: The expression of CXCL13 in human serum, prostate tissue and cell lines. ( A ) Serum CXCL13 concentration in BPH patients and healthy controls. ( B , C ) The protein and mRNA expression of CXCL13 in BPH tissues and normal ones. ( D – F ) The mRNA and protein expression of CXCL13 in BPH-1 and WPMY-1 cells. Representative blots are shown. * p < 0.05, ** p < 0.01 and *** p < 0.001. ( G , H ) Immunofluorescence localization of CXCL13 in BPH tissues and normal ones. ( I , J ) Immunofluorescence localization of CXCL13 in BPH-1 and WPMY-1 cells. DAPI (blue) indicates nuclear staining and Cy3-immunofluorescence (red) indicates CXCL13 protein staining. Representative graphs are shown. All scale bars are 100 μm.

Article Snippet: Human benign prostatic enlargement epithelia cell line BPH-1 (Cat. BNCC339850) was purchased from the Procell Co., Ltd. in Wuhan, China.

Techniques: Expressing, Concentration Assay, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: rHuCXCL13 treatment promoted proliferation of BPH-1 cells, fibrosis and inflammation of WPMY-1 cells. ( A , B ) Flow cytometry analysis of cell cycle and cell apoptosis in BPH-1 cells. ( C , D ) Statistical analysis of percentages (%) of cells at each stage and apoptotic rate (%) in BPH-1 cells. ( E ) CCK8 assay of BPH-1 cells. ( F , G ) Western blot assay of EMT-, cell-cycle- and apoptosis-related proteins in BPH-1 cells. ( H – J ) The mRNA and protein expression of markers of fibrosis and inflammation in WPMY-1 cells. * p < 0.05, ** p < 0.01 and ns means no significant difference.

Article Snippet: Human benign prostatic enlargement epithelia cell line BPH-1 (Cat. BNCC339850) was purchased from the Procell Co., Ltd. in Wuhan, China.

Techniques: Flow Cytometry, CCK-8 Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: Knockdown of CXCL13 inhibited proliferation, EMT and promoted apoptosis of BPH-1 cells via ERK1/2 and AKT pathway. ( A – C ) Knockdown efficiency of CXCL13 at mRNA and protein levels. ( D , F ) Statistical analysis of apoptotic rate (%) and percentages (%) of cells at each stage. ( E , G ) Flow cytometry analysis of cell apoptosis and cell cycle. ( H ) CCK8 assay of BPH-1 cells. *** means p < 0.001 between si-con and si-2, ### means p < 0.001 between si-2 and si-2+rHuCXCL13. ( I , J ) Western blot assay of cell-cycle-, cell-apoptosis- and pathway-related proteins. ( K ) Relative densitometric quantification of cell-cycle-, cell-apoptosis- and pathway-related proteins. ( L ) Western blot assay of EMT-related proteins. ( M ) Relative densitometric quantification of EMT-related proteins. * p < 0.05, ** p < 0.01, *** p < 0.001 and ns means no significant difference.

Article Snippet: Human benign prostatic enlargement epithelia cell line BPH-1 (Cat. BNCC339850) was purchased from the Procell Co., Ltd. in Wuhan, China.

Techniques: Knockdown, Flow Cytometry, CCK-8 Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: Anti-CXCR5 (1 μg/mL) could rescue the alternations induced by rHuCXCL13 in BPH-1 cells. ( A , B ) Flow cytometry analysis of cell cycle and cell apoptosis. ( C , D ) Statistical analysis of percentages (%) of cells at each stage and apoptotic rate (%). ( E ) CCK8 assay. ( F – H ) Western blot assay of cell-cycle-, apoptosis-, EMT- and pathway-related proteins. ( I ) Relative densitometric quantification of cell-cycle-, apoptosis-, EMT- and pathway-related proteins. * p < 0.05, ** p < 0.01, *** p < 0.001 and ns means no significant difference.

Article Snippet: Human benign prostatic enlargement epithelia cell line BPH-1 (Cat. BNCC339850) was purchased from the Procell Co., Ltd. in Wuhan, China.

Techniques: Flow Cytometry, CCK-8 Assay, Western Blot