study zr 75 1  (ATCC)

Journal: British Journal of Cancer

Article Title: Nuclear CSPP1 expression defined subtypes of basal-like breast cancer

doi: 10.1038/bjc.2014.297

Figure Lengend Snippet: Differential expression and localisation of CSPP1 proteins in human breast cancer cell lines. ( A ) Low-magnification images of immunofluorescence stained luminal type (ZR-75-1, MCF7, UACC-812, BT-474) and basal-like (HCC1937, MDA-MB-231) breast cancer cell lines with the common CSPP and CSPP-L C-terminus directed antibody (a-CSPP/CSPP-L) and a CSPP-L-specific antibody (a-CSPP-L) shows nuclear a-CSPP/CSPP-L staining in luminal type cancer cell lines. Individual channels (a-CSPP-L, green; a-CSPP/CSPP-L, red; DNA, blue) and a colour merged image are shown for each cell line. Images were acquired at identical imaging settings and are presented at identical contrast scales. ( B ) High-magnification imaging shows centrosomal co-localisation of a-CSPP/CSPP-L staining (red) and a-Pericentrin (centrosome, green) and ( C ) co-staining of a-CSPP/CSPP-L (red) and a-CSPP-L (green) at the mitotic spindle (ZR-75-1), centrosomes and cytokinetic bridge microtubules (HCC1937). ( D ) a-CSPP-L (green) staining is also detected at primary cilia (a-glutamylated tubulin, red) in MCF10A and MDA-MB-231, which differs in ciliation frequency and cilia morphology.

Article Snippet: Breast cancer cell lines used in this study (MCF7, ZR-75-1, BT-474, UACC-812, HCC1937, HCC38, MDA-MB-231, MCF10A), are of the authenticated ATCC ICBP-43 Breast Cancer Panel and were cultivated according to ATCC's subculturing procedures (#30-4500 K, ATCC, Manassas, VA, USA).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining, Imaging

Journal: British Journal of Cancer

Article Title: Nuclear CSPP1 expression defined subtypes of basal-like breast cancer

doi: 10.1038/bjc.2014.297

Figure Lengend Snippet: Characterisation of nuclear CSPP1 protein expression. ( A ) Transient transfection of endogenous nuclear CSPP1 expressing ZR-75-1 cells with selected shRNA and eGFP co-expressing plasmids proof nuclear CSPP1 staining specificity of the a-CSPP/CSPP-L antibody (red). Bar diagram shows quantification of nuclear CSPP1 staining intensity in control and CSPP1 targeting shRNA transfectants co-expressing eGFP (error bars depict s.e.m from three independent experiments (300 eGFP-positive cells/experiment). ( B ) a-CSPP-L immunoblotting of total cell lysates from Hek293T cells transiently transfected with plasmids expressing distinct CSPP1 mRNA targeting shRNAs or a control shRNA. Co-expression of eGFP allowed flowcytometric sorting of transfectants and identifies CSPP1 targeting shRNAs with highest knockdown efficacy compared with control protein γ -tubulin. ( C ) Ectopically expressed Myc-tag fusion proteins of CSPP or CSPP-L do not localise to the nucleus of ZR-75-1 cells, but show centrosomal and cytoskeletal staining (a-myc, red; a-Pericentrin, green). ( D ) The ectopically expressed eGFP-taged common C-terminal domain of CSPP/CSPP-L (CSPP 498–876 -eGFP, green) shows nuclear localisation in ZR-75-1 and HCC1937 cells and comprises the a-CSPP/CSPP-L target moiety. ( E ) Nuclear CSPP1 protein expression (a-CSPP/CSPP-L, red) is detected throughout interphase in ZR-75-1 cells, as evaluated by co-staining of cyclin A (green; devoid in G 1 phase, increasing nuclear expression in S-phase and high nuclear and cytoplasmic expression in G 2 phase). DNA is counterstained in all immunofluoresence experiments by Hoechst 33258 and depicted in blue.

Article Snippet: Breast cancer cell lines used in this study (MCF7, ZR-75-1, BT-474, UACC-812, HCC1937, HCC38, MDA-MB-231, MCF10A), are of the authenticated ATCC ICBP-43 Breast Cancer Panel and were cultivated according to ATCC's subculturing procedures (#30-4500 K, ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, shRNA, Staining, Control, Western Blot, Knockdown