Elevated S100A6 (Calcyclin) enhances tumorigenesis and suppresses CXCL14-induced apoptosis in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is often resistant to existing therapy. We found elevated S100A6 levels in ccRCC tissues, associated with higher grade pathological features and clinical stages in ccRCC patients. Knockdown of S100A6 inhibited cell proliferation in vitro and tumor growth in vivo. Gene expression profiling suggests a novel function of S100A6 in suppressing apoptosis, as well as a relationship between S100A6 and CXCL14, a pro-inflammatory chemokine. We suggest that the S100A6/CXCL14 signaling pathway is a potential therapeutic target in ccRCC.


Protein extraction and western-blot analysis
Protein levels were quantified by Bradford assay. 30 mg protein from each sample was fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (PVDF membranes, Millipore). The membrane was blocked in 0.1% Triton X-100 and 5% low fat milk powder in phosphate-buffered saline for 1 hour at 4°C and then probed with mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; Abcam, ab9485), mouse monoclonal primary antibody, anti-Calcyclin(F-1) (1:500, Santa Cruz Biotechnology, sc-271396), rabbit polyclonal primary antibody, anti-CXCL14 (1:1000, Abcam, ab46010). After washing 3 times with Tris-buffered saline Tween-20, the membrane was incubated in peroxidase-conjugated goat anti-mouse/ rabbit IgG antibody (1:1500, Santa Cruz Biotechnology). Bands were visualized by an enhanced chemiluminescence detection system using medical X-ray films and quantified by Photoshop (Adobe software). The intensities of band of interest were expressed relative to the GAPDH intensities from the same sample.

Immunohistochemistry
All samples were fixed in 10% neutral formalin. Sections were cut from wax blocks and mounted on APES-coated glass slides. Slides were dewaxed in xylene twice for 10 min and rehydrated in a graded ethanol series. Antigen retrieval was performed in 0.01 mol/L citrate buffer (Ph 6.0) by microwave oven for 2 min and 30 sec at 100°C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. After washing with phosphate buffered saline (PBS), sections with mouse monoclonal primary antibody, anti-Calcyclin(F-1) (1:200, Santa Cruz Biotechnology, sc-271396) in blocking solution was incubated at 4°C overnight in a humidified chamber. After washing three times with PBS, sections were incubated for 30 min with biotinylated secondary antibody (monoperoxidase-anti-mouse IgG, ZSGB-BIO). After washing in PBS 3 times, 3'-diaminobenzidine (DAB) was used as the chromogen. Slides were counterstained with hematoxylin for 5 min. Images were acquired with an OLYMPUS IX81 microscope (OLYMPUS, Japan). The images of tumor sections were taken at 100 × and 200 × magnification.

Cell proliferation assay
After treatment, the cells were plated in 96-well plates at a density of 2500 cells/well. On the 0, 24, 48, 72, 96 h of culture, the cells were washed and subjected to a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega, USA), which was read at a wavelength of 490 nm using a 96-well ELx800TM Absorbance Microplate Reader (BiotekR, USA). Background 490 nm absorbance from the non-cellular control wells containing the same volumes of culture medium and CellTiter 96 ® AQueous One Solution Reagent was subtracted. All analyses were performed in triplicate on separate occasions. The mean and standard error at 490 nm absorbance of the experimental and control groups were analyzed at each of the aforementioned time points.

Cell cycle and apoptosis
The stable transfected cells were collected, and treated with 70% ethanol at 4°C overnight, and then washed with ice-cold PBS. For the detection of cell cycle, cell pellets were resuspended in RNase-containing (1:100 in dilution)PBS buffer in ice, and stained with propidium iodide (PI) (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Stained cells were analyzed on the FACS-Calibur (BD Biosciences). Data were analyzed using the Cellquest Pro software (BD Biosciences). Flow cytometry was performed to detect apoptosis with

Oncotarget, Supplementary Materials 2015
AnnixinV-PE apoptosis Detection Kit (Beyotime). Cells were collected and resuspended in 0.5 ml PBS buffer. To these cells, Annixin V-PE were added lucifugal and incubated according protocol, and then analyzed by FCM (FACS-Calibur, Becton Dickinson, San Jose, CA).

RNAi knockdown
Three small interfering RNA (siRNA) duplexes targeting different coding regions of human CXCL14 and their scrambled sequence siRNA (mock) were custom synthesized by Shanghai Gene-Pharma Co. (Shanghai, China). For the RNAi knockdown, equal numbers of cells were seeded in the plates containing medium without antibiotics for 24 h prior to the transfection. The siRNAs were introduced into the cells using Lipofectamine 2000 in serum-free Opti-MEM, according to the manufacturer's instructions. The expression levels of CXCL14 were determined after 72 h by western blot analyses ( Figure 5A). The most efficient siRNA for knockdown was renamed as si-CXCL14 1# and 2#, the si-sequence were shown in Supplementary Table S2, chosen for further experiments, the scrambled sequence siRNA was named as si-scramble. The transfected cells were grown in complete medium at 37°C and 5% CO 2 .The cells were harvested at the indicated time points and used for further analysis.

eGFP Fluorescence
The eGFP fluorescence of the samples was detected and analyzed in vitro using a molecular imaging system (NightOWL II LB 983, Berthold, Germany). The signal intensity of the eGFP fluorescence from the tissues represented the amount of tumor lesions.   (21) All primers for real-time reversion PCR were designed span the exon-exon junction.