serum cypa  (BioVendor Instruments)

Journal: BMC Cancer

Article Title: Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

doi: 10.1186/1471-2407-12-442

Figure Lengend Snippet: Expression of CypA in six lung cancer cell lines and MRC5 cells. A , CypA mRNA expression in five NSCLC cell lines (95C, A549, H460, H1299, and A2), one SCLC cell line (H446) and MRC5. The 95C, A549, H460, H446, H1299, and A2 cells exhibited 12.02-, 5.19-, 4.62-, 3.40-, 2.28-, and 1.94-fold increases, respectively, in CypA mRNA expression compared to MRC5 cells. The relative mRNA level is presented as 2 -ΔCT . All data shown are means ± SEM from three separate experiments. ** P <0.01 (compared with MRC5, t -test). B , CypA protein level in 95C, A549, H1299, A2, H446 and MRC5 cell lines were detected by western-blot, GAPDH was used as internal loading controls (upper panel). CypA protein level was also measured by immunofluorescence staining. The fluorescence intensity was quantified by Cellomics ArrayScan HCS Reader using the ArrayScanTM software (lower panel). The CypA protein level normalized to MRC-5. ** P <0.01 (compared with MRC5, t -test) C , Proliferation of 95C and A549 cells. OD 490 values were determined daily at predetermined time points. The doubling-time of 95C and A549 cells was 24h and 43 h respectively. The data are expressed as the mean ± SEM of triplicate values from three separate experiments. D , Mobility of 95C and A549 cells. The percent distance of wound closure at 24 h following wound generation was calculated from three separate areas and is expressed as the mean±SEM. ** P <0.01 ( t -test).

Article Snippet: The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Software

Journal: BMC Cancer

Article Title: Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

doi: 10.1186/1471-2407-12-442

Figure Lengend Snippet: Interference with CypA expression in 95C and A549 cells. A , CypA mRNA expression in 95C and A549 cells with CypA-targeting RNAi. The relative mRNA level is presented as 2 -ΔCT . The data are the means ± SEM of three separate experiments. B , CypA protein levels in 95C and A549 cells with CypA-targeting RNAi. Cell lysates prepared from each group were screened using Western blotting with anti-CypA antibodies. The data are expressed as the mean ± SEM of triplicate values from three separate experiments. ** P <0.01 ( t -test).

Article Snippet: The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6).

Techniques: Expressing, Western Blot

Journal: BMC Cancer

Article Title: Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

doi: 10.1186/1471-2407-12-442

Figure Lengend Snippet: Effect of CypA on tumorigenesis. A , CypA depletion inhibited the proliferation of 95C and A549 cells. OD 490 values were determined daily at predetermined time points. The data are expressed as means ± SEM of triplicate values from three separate experiments. * P <0.05, ** P <0.01 ( t -test). 95C, 95C-MOCK, 95C-KD cell doubling-time was 24h, 26h, 31h respectively. And cell doubling-time of A549, A549-MOCK, A549-KD was 43h, 44h, 49h respectively. B, the effect of 239836 on cell proliferation was detected by MTS assay. The data are expressed as means ± SEM. ** P <0.01 ( t -test) C , CypA-assisted 95C and A549 cells avoided density-dependent growth inhibition. Colony formation after 10 days was quantified by counting the number of colonies per well. The values represent the mean number of colonies±SEM from three separate wells. ** P <0.01 ( t -test). D , The knockdown of CypA suppressed the anchorage-independent proliferation of 95C and A549 cells. Colony formation after 21 days was quantified by counting the number of colonies per well. The values represent the mean number of colonies (the mean±SEM) from three separate wells. ** P <0.01 ( t -test).

Article Snippet: The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6).

Techniques: MTS Assay, Inhibition, Knockdown

Journal: BMC Cancer

Article Title: Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

doi: 10.1186/1471-2407-12-442

Figure Lengend Snippet: The phosphorylation level of ERK1/2 and p38 was deregulated by the suppression of CypA expression. Western-blot analysis of A549, A549 MOCK, A549 KD with antibodies to ERK1/2 ( A ), p38( B ), JAK2( C ), STAT5( D ) and their phosphorylated forms were carried out. GAPDH was used as internal loading controls.

Article Snippet: The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6).

Techniques: Phospho-proteomics, Expressing, Western Blot

Journal: BMC Cancer

Article Title: Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

doi: 10.1186/1471-2407-12-442

Figure Lengend Snippet: The rates of migration and invasion in CypA interfered 95C and A549 cells with A, Migration was investigated using a wound healing assay. The percent areas of wound closure at 24 h following wound generation were calculated from three separate areas and are expressed as the mean±SEM. ** P <0.01 ( t -test). Photomicrographs were taken after 24 h (original magnification, ×100). B , Other migration assays were performed using a Transwell unit. Cells on the underside of the insert filters were fixed, stained, and counted under a microscope. The data are expressed as means ± SEM of triplicate values from three separate experiments. ** P <0.01 ( t -test). Photomicrographs were taken after 24 h (original magnification, ×200). C , The in vitro invasive properties of 95C and A549 cells was tested using a Matrigel-coated Transwell unit. Cells on the underside of the insert filters were fixed, stained, and counted under a microscope. The data are expressed as means ± SEM of triplicate values from three separate experiments. ** P <0.01 ( t -test). Photomicrographs were taken after 48 h (original magnification, ×400).

Article Snippet: The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6).

Techniques: Migration, Wound Healing Assay, Staining, Microscopy, In Vitro

Journal: BMC Cancer

Article Title: Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

doi: 10.1186/1471-2407-12-442

Figure Lengend Snippet: Zymographic analysis of MMP9 and MMP2 activity in 95C cells. A , Conditioned media prepared from each cell group were screened using gelatin zymographic analysis. The densities of MMP9 were determined and plotted. The data are expressed as the means ± SEM of triplicate values from three separate experiments. ** P <0.01( t -test). B , Conditioned media prepared from CypA inhibitor 239836 treated cells as well as control group were screened using gelatin zymographic analysis. The gels were scanned and analyzed using Alpha View Analysis Tools. * P <0.05.

Article Snippet: The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6).

Techniques: Activity Assay, Control