human prostate tumor derived lncap cells  (ATCC)

Journal: Scientific Reports

Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

doi: 10.1038/s41598-017-02918-3

Figure Lengend Snippet: Androgen deprivation and AR inhibition result in increased CXCR7 mRNA transcription in androgen-responsive prostate cancer cells. ( a ) CXCR7 mRNA abundance in LNCaP, C4-2B, and 22Rv1 cell lines relative to PPIA. ( b ) CXCR7 mRNA transcription in androgen deprived cells relative to androgen stimulated cells. ( c ) CXCR7 mRNA in cells treated with 2 µM bicalutamide (BiC) relative to untreated (UT) cells. ( d ) CXCR7 mRNA in cells treated with 5 µM enzalutamide (Enz) relative to UT cells. All graphs shown include the means of 3 independent experiments, ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ns = no significance; unpaired, two-tailed Student’s t-test.

Article Snippet: Human prostate epithelial tumor cell lines LNCaP (American Type Culture Collection [ATCC]; Manassas, VA; CRL-1740) and CRW-22Rv1 (ATCC; CRL-2505) were cultured in RPMI-1640 (Corning cellgro; Corning, NY; 10-040-CV), and C4-2B cells (ViroMed Laboratories; Burlington, NC; 12–103) were cultured in T-medium prepared as described previously ; media were supplemented with 10% (5% for T-medium) fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA) and 10 μg/mL gentamicin (Sigma-Aldrich; St. Louis, MO).

Techniques: Inhibition, Two Tailed Test

Journal: Scientific Reports

Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

doi: 10.1038/s41598-017-02918-3

Figure Lengend Snippet: AR regulates CXCR7 expression and downstream EGFR-mediated mitogenic signaling. ( a ) Left: western blot panel of indicated proteins from LNCaP cells treated in the presence or absence of 5 nM R1881, followed by 2 or 5 minutes of EGF stimulation. Right: Densitometry analysis of phosphorylated protein relative to total; and AR or CXCR7 relative to actin. ( b ) Western blot panel of indicated proteins from LNCaP cells transfected with control siRNA (siCtl) or AR siRNA (siAR) treated with vehicle (DMSO) or 5 nM R1881. ( c ) CXCR7 mRNA expression in siCtl or siAR transfected cells with or without R1881 stimulation. n = 3, mean ± SEM; ***p < 0.001, ns = no significance; one-way ANOVA, secondary Tukey’s multiple comparisons test. The western blots shown in a and b are representative of three independent experiments with similar outcomes; densitometry is measured from the western blot panel shown.

Article Snippet: Human prostate epithelial tumor cell lines LNCaP (American Type Culture Collection [ATCC]; Manassas, VA; CRL-1740) and CRW-22Rv1 (ATCC; CRL-2505) were cultured in RPMI-1640 (Corning cellgro; Corning, NY; 10-040-CV), and C4-2B cells (ViroMed Laboratories; Burlington, NC; 12–103) were cultured in T-medium prepared as described previously ; media were supplemented with 10% (5% for T-medium) fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA) and 10 μg/mL gentamicin (Sigma-Aldrich; St. Louis, MO).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Scientific Reports

Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

doi: 10.1038/s41598-017-02918-3

Figure Lengend Snippet: CXCR7 expression is directly regulated by AR binding to the CXCR7 promoter. ( a ) CXCR7 promoter-driven luciferase activity in C4-2B cells cultured in CDFBS ± R1881, relative to untreated control cells (n = 3, mean ± SEM). ( b ) Diagram of the cloned CXCR7 promoter region including the 6 putative AREs tested for AR binding. ( c ) ChIP assay for AR pulldown of promoter regions shown in B in LNCaP ± R1881. The PSA enhancer region served as a positive control for AR binding. n = 3, mean ± SEM; *p < 0.05, ***p < 0.001, ns = no significance; unpaired, two-tailed Student’s t-test.

Article Snippet: Human prostate epithelial tumor cell lines LNCaP (American Type Culture Collection [ATCC]; Manassas, VA; CRL-1740) and CRW-22Rv1 (ATCC; CRL-2505) were cultured in RPMI-1640 (Corning cellgro; Corning, NY; 10-040-CV), and C4-2B cells (ViroMed Laboratories; Burlington, NC; 12–103) were cultured in T-medium prepared as described previously ; media were supplemented with 10% (5% for T-medium) fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA) and 10 μg/mL gentamicin (Sigma-Aldrich; St. Louis, MO).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Cell Culture, Control, Clone Assay, Positive Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

doi: 10.1038/s41598-017-02918-3

Figure Lengend Snippet: CRISPR-Cas9-mediated frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the CXCR7-gRNA target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).

Article Snippet: Human prostate epithelial tumor cell lines LNCaP (American Type Culture Collection [ATCC]; Manassas, VA; CRL-1740) and CRW-22Rv1 (ATCC; CRL-2505) were cultured in RPMI-1640 (Corning cellgro; Corning, NY; 10-040-CV), and C4-2B cells (ViroMed Laboratories; Burlington, NC; 12–103) were cultured in T-medium prepared as described previously ; media were supplemented with 10% (5% for T-medium) fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA) and 10 μg/mL gentamicin (Sigma-Aldrich; St. Louis, MO).

Techniques: CRISPR, Activity Assay, Mutagenesis, Expressing, Negative Control, Positive Control, Sequencing

Journal: Scientific Reports

Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

doi: 10.1038/s41598-017-02918-3

Figure Lengend Snippet: CRISPR-Cas9-generated CXCR7 inframe mutation in LNCaP cells disrupts membrane protein interactions. ( a ) Left: PCR of CXCR7 genomic DNA in unmodified cells (WT) and CRISPR-edited LNCaP cells revealing a 394 nt deletion event (CX7-mutant). Right: Sanger sequencing chromatogram of the DNA sequence illustrating the contiguous sequence, Black arrow represents the expected 5′ CXCR7 sequence and the red arrow shows the 3′ CXCR7 sequence around the site of deletion event. ( b ) EGFR and ERK phosphorylation in WT and CX7-mutant cells with densitometry of phosphorylated protein relative to total. ( c ) Count of second generation spheroids formed per plate from 10,000 seeded WT and CX7-mutant cells (n = 2, mean ± SD; *p < 0.05; unpaired, two-tailed Student’s t-test. ( d ) PLA staining for CXCR7 protein interaction with ARRB1, ARRB2, or isotype control in the WT and CX7-mutant cell lines. Nuclei are stained with DAPI (blue); red spots indicate that the two target proteins are co-localized within 40 nm of one another. The western blots are representative of three independent experiments with similar outcomes; densitometry is measured from the western blot panel shown.

Article Snippet: Human prostate epithelial tumor cell lines LNCaP (American Type Culture Collection [ATCC]; Manassas, VA; CRL-1740) and CRW-22Rv1 (ATCC; CRL-2505) were cultured in RPMI-1640 (Corning cellgro; Corning, NY; 10-040-CV), and C4-2B cells (ViroMed Laboratories; Burlington, NC; 12–103) were cultured in T-medium prepared as described previously ; media were supplemented with 10% (5% for T-medium) fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA) and 10 μg/mL gentamicin (Sigma-Aldrich; St. Louis, MO).

Techniques: CRISPR, Generated, Mutagenesis, Membrane, Sequencing, Phospho-proteomics, Two Tailed Test, Staining, Control, Western Blot