human normal prostate epithelial cells rwpe 1  (Procell Inc)

Journal: The World Journal of Men's Health

Article Title: Inhibition of Ferroptosis in Prostatitis Model by Low Intensity Extracorporeal Shock Wave Therapy through the Integrin-β1/NRF2 Axis

doi: 10.5534/wjmh.250222

Figure Lengend Snippet: Integrin-β1 served as a key mechanoreceptor mediating the anti-ferroptotic action of Li-ESWT. (A–E) Western blot analysis of Integrin-β1, NRF2, and the ferroptosis-related proteins xCT and GPX4 in RWPE-1 cells was performed to evaluate the effect of Li-ESWT-mediated Integrin-β1 activation on NRF2-xCT/GPX4 axis. (F–I) Lipid peroxidation and intracellular iron levels were assessed by flow cytometry using the C11 BODIPY and RhoNOX-6 probes, respectively, to evaluate the occurrence of ferroptosis following Li-ESWT, Integrin-β1 knockdown, or ferroptosis inhibitor Fer-1 treatment. Data were presented as mean±standard deviation. Li-ESWT: low-intensity extracorporeal shock wave therapy, Fer-1: Ferrostatin-1, LPS: lipopolysaccharide. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Human normal prostate epithelial cells RWPE-1 (Procell) were cultured in Prostate Epithelial Cell Medium (ScienCell).

Techniques: Western Blot, Activation Assay, Flow Cytometry, Knockdown, Standard Deviation

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 2. Knocking down PLAC8 expression inhibits Cd-induced tumor growth in xenotransplanted mice. (A) In CTPE cells, silencing PLAC8 expression reduced tumor formation in the xenotransplantation model. (B) Immunohistochemistry (IHC) of tumor tissues analyzed for Ki-67, PLAC8, LC3b, and LAMP1 expression. (C) A volcano plot analysis displayed the differential expression of genes in sh-PLAC8 tumors compared to the control group. (D) GSEA identified pathways associated with prostate cancer, lysosomal functions, and NF-κB–mediated TNF-α signaling in PLAC8-knockdown (PLAC8_KD) tumors compared to the vector control. (E) Cd-transforming cells showed a time-dependent induction of p65 expression (F) and NF-κB activation was observed. (G) Both cytosolic and nuclear expression of p65 were noted during the transforma- tion of Cd-exposed RWPE-1 cells. (H) p65 binding sites on the PLAC8 promoter were identified and validated by comparing luciferase activity in wild-type and mutated (Δ) sites, transcription start sites (TSS) and (I) ChIP-qPCR was performed in CTPE cells. All error bars represent means ± SD, with statistical significance indicated as *P < 0.05, ***P < 0.001; ns, not significant. NES, normalized enrichment score.

Article Snippet: MATERIALS AND METHODS Cell lines and reagents Human normal prostate epithelial (RWPE- 1) cells were purchased from the American Type Culture Collection (VA, USA) and subcultured with the recommended protocol.

Techniques: Expressing, Immunohistochemistry, Quantitative Proteomics, Control, Knockdown, Plasmid Preparation, Activation Assay, Binding Assay, Luciferase, Activity Assay, ChIP-qPCR

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 3. The interaction between PLAC8 and NF-κB during the transformation of prostate epithelial cells. (A) The interaction between p65 and PLAC8 is confirmed by immunoprecipitation (IP) analysis. IgG, immunoglobulin G. (B) CHX was used to inhibit protein synthesis in vector alone and sh-p65 cells, and Western blot (WB) analysis was performed to show that p65 is necessary to stabilize PLAC8 in CTPE cells. h, hours. (C) Immunofluorescence of RWPE-1 and Cd-transforming cells shows an increase in percent- age of cells with PLAC8 and p65 colocalization. (D) Immunofluorescence staining and the colocalization analysis of p65 and PLAC8 were assessed using Pearson coefficient. (E) Ectopic expression of p65 increases PLAC8 expression in RWPE-1 cells. (F) The expression levels of p65, PLAC8, LAMP1, and LC3B were determined by Western blot analysis in sh-p65–transfected cells, both in the presence and absence of Cd. (G) Immunofluorescence staining and colocalization analysis of LC3B and LAMP1 fusion with increased Pearson coefficient in sh-p65 CTPE cells. (H) Representative TEM images showing fusion of autophagosomes and lysosomes in sh-p65–transfected CTPE cells compared to vector alone, along with the quantification of autophagosomes, lysosomes, and autolysosomes per square micrometer. Arrowheads indicate lysosomes (in blue), autophagic vacuoles (in red), and autolysosomes (in green). All error bars represent means ± SD. Statistical significance is indicated as *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: MATERIALS AND METHODS Cell lines and reagents Human normal prostate epithelial (RWPE- 1) cells were purchased from the American Type Culture Collection (VA, USA) and subcultured with the recommended protocol.

Techniques: Transformation Assay, Immunoprecipitation, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Expressing, Transfection

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 5. BCL-xL plays a crucial role in the survival of transformed cells and is regulated by PLAC8. (A) Inhibiting the expression of p65 and PLAC8 enhances the induc- tion of apoptosis in CTPE cells, confirmed by flow cytometry analysis of annexin V-FITC–stained apoptotic cells. (B) Ectopic expression of PLAC8 leads to increased levels of BCL-xL and p65 in RWPE-1 cells. (C) The expression of BCL-xL is observed at successive stages of Cd exposure during the transformation of RWPE-1 cells. (D) Silencing BCL-xL expression abolishes the PLAC8-mediated autophagy signaling in CTPE cells. (E) Ectopic expression of BCL-xL results in up-regulating PLAC8 and p65 in RWPE-1 cells. (F) In CTPE cells, cotransfection with sh-PLAC8 and the pCMV p65 overexpression plasmid demonstrated PLAC8, p65, and BCL-xL protein levels through Western blot analysis. (G) A luciferase assay showing increased BCL-xL promoter activity in CTPE cells compared to RWPE-1 cells. All error bars represent means ± SD, with statistical significance at *P < 0.05.

Article Snippet: MATERIALS AND METHODS Cell lines and reagents Human normal prostate epithelial (RWPE- 1) cells were purchased from the American Type Culture Collection (VA, USA) and subcultured with the recommended protocol.

Techniques: Transformation Assay, Expressing, Flow Cytometry, Staining, Cotransfection, Over Expression, Plasmid Preparation, Western Blot, Luciferase, Activity Assay