Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Summary of genes methylated within the non-invasive LNCaP and DU145 cell lines

Article Snippet: Primary human prostate cancer cells (PCSC1-2) [ ] were acquired from Celprogen (San Pedro, CA) and maintained as recommended using specific coated culture plates and defined media.

Techniques: Methylation

Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Oncomine analysis of overlapping targets methylated in both LNCaP and DU145 cells . Isolated targets from the methylation arrays overlapping in LNCaP and DU145 cells were analyzed in Oncomine 4.2 (Ann Arbor, MI). The heat map represents raw data from the Varambally over-expression in prostate cancer analysis comparing primary tissue and metastatic tissue . Expression is in terms of normalized over-expression units. The P-value represents a student's t-test comparing primary and metastatic expression and the gene ID is provided. Genes of interest included Sox1 (p = 0.024) since it has high homology to the stem cell gene Sox2 and albeit demonstrating significance, Bmx (p = 0.294), since it has previously been implicated in prostate cancer regulation.

Article Snippet: Primary human prostate cancer cells (PCSC1-2) [ ] were acquired from Celprogen (San Pedro, CA) and maintained as recommended using specific coated culture plates and defined media.

Techniques: Methylation, Isolation, Over Expression, Expressing

Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Validation of methylated targets in LNcaP and DU145 cells . A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.

Article Snippet: Primary human prostate cancer cells (PCSC1-2) [ ] were acquired from Celprogen (San Pedro, CA) and maintained as recommended using specific coated culture plates and defined media.

Techniques: Methylation, Modification, Agarose Gel Electrophoresis, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay, Staining, Incubation, Microscopy

Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Functional Role of SOX1 during invasion . A) The Trans-Lentiviral pTRIPZ system from Open Biosystems was used to introduce shRNA against BMX, SOX1 or a non-silencing control vector in DU145 cells. The cells were selected for 2 weeks in 1 μg/mL of puromycin and single cell clones were generated. To induce expression of the shRNA 1 μg/mL of doxycycline was added. The plasmid is designed to have a TET inducible TurboRFP upstream of the shRNA and they should appear red upon successful infection. B) Lowered expression was confirmed using Western blotting. Follow up experiments were conducted using BMX clone 3 and 5 and SOX1 clone 7 and 8 since they demonstrated the most significant decrease in protein expression. Fold changes represent samples normalized to actin and the control level of expression. C) Proliferation assays were conduced using Cell Titer-Glo kit and assayed on Day 1, 3, 5 and 7. More proliferation is indicated by an increase in relative luciferase units (RLUs). *denotes statistical significant p < 0.05 compared to vector transfected cells. A significant decrease was observed in shSOX1 #7 cells compared to vector transfected cells, and a significant increase was observed in shBMX #5 cell line. D) Matrigel invasion assays were conducted for 24 hours toward SCM. Top cells were removed and bottom cells were stained with the Diff-Quick staining kit from Dade Behring. Cells were counted using 4 independent fields per sample and 2 chambers were used per cell line. *denotes statistical significance p < 0.05 compared to vector transfected cells. Both shSOX #7 and #8 demonstrated significant decreases in invasion toward SCM compared to vector transfected cells.

Article Snippet: Primary human prostate cancer cells (PCSC1-2) [ ] were acquired from Celprogen (San Pedro, CA) and maintained as recommended using specific coated culture plates and defined media.

Techniques: Functional Assay, Introduce, shRNA, Plasmid Preparation, Clone Assay, Generated, Expressing, Infection, Western Blot, Luciferase, Transfection, Staining, Diff-Quik

Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Direct interaction between SOX1 and STAT3 . A) Matrigel invasion assays were performed for 24 hours toward SCM using DU145 cells in the presence of the anti-IL-6, the PI3K inhibitor LY294002, a small molecular inhibitor of MEK called U0126 (thus downstream inhibition of extracellular-related kinase (ERK1 and ERK2) mediated responses), a small molecule inhibitor JAK called AG490 (Janus Kinase) and an inhibitor of its partner signal transducers and activators of transcription-3 (STAT3) called Static or the Tec kinase family inhibitor LFM-A13.Significant differences were observed between control cells and those cells treated with U0126, Stattic, LY294002 and LFM-A13. B) qRT-PCR analysis was performed as mentioned in Figure 3. *denotes statistical significant p < 0.05 compared to the non-invasive cells. Increased levels of Stat3 are seen invasive LNCaP and DU145 cells compared to the parental lines. C) Staining of pSTAT3 in invasive or non-invasive DU145 cells was performed directly on the Matrigel membrane and carried out as previously described in Figure 3. D) DU145 lysates were incubated with either SOX1, STAT3 or BMX overnight at 4°C with rotation. Samples were then incubated with Protein A-agarose beads to isolate complexes. Membranes were then incubated overnight at 4°C using primary antibodies for STAT3 or SOX1. The membrane was developed using the Odyssey from Licor. Protein loading was normalized using actin as a control. E) Western blotting for STAT3 and pSTAT3 in sub-cellular protein extracts from DU145, NS, shBMX#3 or shSOX1#8. F) STAT3 EMSA: Each lane contains WT-IR STAT3 oligos. Lane 1-3 DU145, 4 and 5 NS, 6 and 7 shBMX#3, 8 and 9 shSOX1 #8 and 10 contains no protein. Lane 2 contains excess MU-IR STAT3 and lanes 3, 5, 7 and 9 contain excess unlabeled WT probe. Supershifited samples appear below and only contain WT-IR STAT3.

Article Snippet: Primary human prostate cancer cells (PCSC1-2) [ ] were acquired from Celprogen (San Pedro, CA) and maintained as recommended using specific coated culture plates and defined media.

Techniques: Inhibition, Quantitative RT-PCR, Staining, Incubation, Western Blot

Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Inhibitor studies further determine that the IL-6/STAT3 pathway is involved in invasion . A) qRT-PCR demonstrating decreased expression of Stat3 in DU145 shSOX1 clone #7 cells and Mcl-1 , a Stat3 target gene. No change was observed in Myc or Survivin . *denotes statistical significant p < 0.05 compared to the vector transfected line. B) Prostatospheres were generated by culturing LNCaP cells in SCM+KO for 7 days and qRT-PCR analysis was performed. Compared to adherent LNCaP cells, expression of Bmx, Sox1 Mcl-1, Myc, Survivin , and Stat3 was significantly increased in the stem-like prostatospheres. *denotes statistical significant p < 0.001 compared to the adherent cells. C) Correlation of Sox1 and Stat3 was analyzed in Oncomine 4.2 (Ann Arbor, MI). The heat map represents raw data from the Varambally over-expression in prostate cancer analysis comparing primary tissue and metastatic tissue. Expression is in terms of normalized over-expression units. The P-value represents a student's t-test comparing primary and metastatic expression. D) Using the GEO database, expression of Sox1 and Bmx were compared between benign, primary or metastatic prostate tissue and significant differences were observed in Sox1 . For Stat 3, a comparison between Prostate Intraepithelial Neoplasia (PIN) and invasive prostate tumor tissue yield a significant difference in expression. *denotes statistical significant p < 0.05 compared to either benign samples or PIN.

Article Snippet: Primary human prostate cancer cells (PCSC1-2) [ ] were acquired from Celprogen (San Pedro, CA) and maintained as recommended using specific coated culture plates and defined media.

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Generated, Over Expression

Journal: bioRxiv

Article Title: Salvianolic acids are natural senolytics and increase lifespan in old age

doi: 10.64898/2026.04.29.721790

Figure Lengend Snippet: ( a ) A schematic workflow illustrating the preclinical therapeutic procedure. Two weeks after subcutaneous inoculation and in vivo uptake of PC3/PSC27 recombinants, severe combined immunodeficient (M-NSG) mice were subject to either single agent or combinatorial treatment in a metronomic schedule of several cycles. The illustration was created in BioRender. ( b ) Comparative statistics of tumor end volumes. PC3 cancer cells were inoculated either alone or combined with PSC27 stromal cells before being implanted subcutaneously to the hind flank of M-NSG animals, which were administered with MIT and SAA, either alone or in combination. ( c ) Representative images of in vivo senescence in tumor foci by SA-β-gal staining. Scale bar, 50 μm. ( d ) Comparative statistics of tumor senescence as described in ( c ). ( e ) Transcriptional analysis of a subset of SASP factors expressed in epithelial vs. stromal cells acquired from tumor foci after laser capture microdissection (LCM) to isolate stromal and cancer cells, respectively. Signals were normalized to that of the sample with lowest value in the placebo group. ( f ) Transcriptional analysis of SASP factors and two canonical senescence biomarkers p16 INK4a and p21 CIP1 . ( g ) Statistical assessment of DDR and cellular apoptosis in tumors. Values are shown as the percentage of cells positively stained by IF or IHC specific to γH2AX or cleaved caspase 3 (CCL3), respectively. ( h ) Representative images of IHC staining for CCL3 at the completion of treatment regimens. Scale bar, 50 μm. ( i ) Comparative survival of animals sacrificed upon development of advanced bulky disease. Survival duration was calculated from tissue recombinant injection until death. P values were calculated by a two-sided log-rank (Mantel-Cox) test. DDR, DNA damage response. IF, immunofluorescence. IHC, immunohistochemistry. Data in b , d , e , f and g are shown as mean ± SD and representative of 3 independent biological replicates, with P values calculated by Student’s t -tests. ^, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The human prostate epithelial cancer cell line, PC3, was from ATCC and cultured with F-12K media (10% FBS).

Techniques: In Vivo, Staining, Laser Capture Microdissection, Immunohistochemistry, Recombinant, Injection, Immunofluorescence