Journal: Cancers

Article Title: The Impact of Cand1 in Prostate Cancer

doi: 10.3390/cancers12020428

Figure Lengend Snippet: ( A ) Statistical analyses and representative Western blot images of Cand1 levels of LNCaP and PC3 cells (fold change of Cand1 expression normalized to GAPDH as loading control) transfected with 50 nM siCtrl or siCand1 and incubated for 96 h. Statistical analysis of cell viability ( B ) and proliferation ( C ) after Cand1 downregulation compared to siCtrl. Data represent mean + SEM from 4 ( A ) or 7 ( B, C ) independent experiments; * p < 0.05, ** p < 0.01. Significance assessed by non-parametric Mann-Whitney U test; ( D ) Representative microscopic pictures (magnification 40×, scale bar 500 µm) of reduced cell number upon Cand1 downregulation in LNCaP and PC3 cells; ( E ) Statistical analyses and representative Western blot images of cPARP levels in LNCaP and PC3 transfected with 50 nM siCtrl or siCand1 and incubated for 96 h. The graph displays fold change of cPARP expression normalized to GAPDH; ( F ) Statistical analysis of caspase 3 and 7 activities in LNCaP and PC3 upon downregulation of Cand1. Statistical analysis of caspase 3/7 activity is shown. The graph displays the percentage of caspase 3 and 7 activities (RFU) normalized to the amount of protein (µg). Caspase-Glo 3/7 assay was carried out 96 h after transfection of LNCaP and PC3 with 50 nM siCtrl or siCand1. Data represent mean + SEM from 5 independent experiments; n.s.: not significant, * p < 0.05. ( G ) Statistical analysis of p21 protein levels after Cand1 downregulation compared to siCtrl. Data represent mean + SEM from 7 ( E, F ) or 4 ( G ) independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001. Significance assessed by non-parametric Mann-Whitney U test; ( H–I ) LNCaP ( H ) and PC3 ( I ) cells were transfected with 50 nM siCtrl (Control), 50 nM siCand1 or 4 µg of pCMV6-Entry-Cand1 overexpression vector and incubated for 72 h. Cand1 was detected using Cand1 RmAb and visualized using goat anti-rabbit secondary antibody (Alexa Fluor 555). Primary antibody anti-KRTB CpAb against Cytokeratin-8 reacted with goat anti-chicken secondary antibody (Alexa Fluor 488). Rabbit mAb IgG served as isotype control. Cell nuclei/DNA were counterstained using DAPI. n = 3. Magnification 400×, scale bar: 50 µm.

Article Snippet: Again, cells were then rinsed four times in PBS and twice in aqua dest., dried on a paper towel and embedded using VECTASHIELD Antifade Mounting Medium containing DAPI (Linaris (VectorLabs), Szabo Scandic, Vienna, Austria), which stains DNA blue.

Techniques: Western Blot, Expressing, Control, Transfection, Incubation, MANN-WHITNEY, Activity Assay, Caspase-Glo Assay, Over Expression, Plasmid Preparation