Images at 3 μm resolution were generated by the Agilent scanner, and. Feature ...... A_22_P00011692. 5,93204. 5,11276. 1,764525158. N/A. N/A. AF086543.
Rossignoli et al., MSC-delivered soluble TRAIL and Paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma, doi:10.7150/thno.27576
SUPPLEMENTARY MATERIALS AND METHODS Tissue culture PaTu 8988t cells (PaTu) were maintained in DMEM (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) integrated with 5% horse serum (Gibco), 5% FBS, 1% L-glutamine and 1% penicillin/streptomycin (all from Carlo Erba Reagents Srl, Cornaredo, Italy). CFPAC-1 cells were cultured
in
IMDM
(Euroclone
Spa,
Milan,
Italy)
supplemented
with
10%
FBS,
1%
penicillin/streptomycin and 2% L-glutamine (all from Carlo Erba Reagents Srl).
Microarray analysis Labelled cRNA was synthesized from 100 ng of total RNA using the Gene expression low input quick amp labelling Kit (Agilent Technologies, Palo Alto, CA, USA) in the presence of cyanine 3CTP (Perkin-Elmer Life Sciences, Boston, MA, USA). Hybridizations were performed at 65 °C for 17 h in a rotating oven. Images at 3 μm resolution were generated by the Agilent scanner, and Feature Extraction 10.5 software (Agilent Technologies) was used to obtain the microarray raw data. RNA was hybridized on Agilent SurePrint G3 Human Gene Expression v3 8x60K Microarray Kit (#G4851C, Agilent Technologies). Microarray results were analyzed using the Qlucore Omics Explorer software (Qlucore). Percentile normalization was used. Experimental sessions data were grouped for both samples analysed and comparative analyses performed between samples.
Rossignoli et al., MSC-delivered soluble TRAIL and Paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma, doi:10.7150/thno.27576
SUPPLEMENTARY FIGURES Supplementary Figure S1. Nab-Paclitaxel treatment is effective in restoring TRAIL sensitivity in resistant BXPC3 cell line
(A) In vitro combined treatment of sTRAIL-containing supernatant with Nab-PTX. PA clone cells were treated for 24 h with AD-MSC EMPTY derived conditioned medium (“EMPTY CM”) or ADMSC sTRAIL derived conditioned medium (“sTRAIL CM”) or the same with 5 ng/ml PTX (“EMPTY CM+Nab-PTX” and “sTRAIL CM+Nab-PTX”). Death rate was evaluated by PI and Annexin V staining in flow cytometry. Death rate for EMPTY CM was 7.0±0.4%, for EMPTY CM+Nab-PTX was 14.0±1.3%, for sTRAIL CM was 8.4±0.7%, for sTRAIL CM+Nab-PTX was 48.8±2.0% (B) Pretreatment strategy included a 24 h incubation with 5 ng/ml PTX for EMPTY CM+Nab-PTX and sTRAIL CM+Nab-PTX groups. The subsequent treatment was performed as in “A” for a further 24 h. Death rate for EMPTY was 6.9±0.5%, for EMPTY CM+Nab-PTX was 13.9±0.6%, for sTRAIL CM was 11.6±0.6%, for sTRAIL CM+Nab-PTX was 56.2±2.3%. All data are represented as the mean
Rossignoli et al., MSC-delivered soluble TRAIL and Paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma, doi:10.7150/thno.27576
of two independent experiments performed in triplicate. * p
