shKC2
Study on the impact of KHSRP-modulated small RNA transcription in a cell transdifferentiation model: Sample shKC2
Mus musculus 10090 NMuMg cells. shKSRP transfected untreated cells.    Cell cultures and treatments.  Murine immortalized NMuMg cells (obtained from ATCC, no. CRL-1636, Lot no.59227127) were cultured in DMEM plus 10% FBS and 10 ug/ml bovine Insulin (Sigma).    HEK-293 cells (obtained from ATCC) were cultured in DMEM plus 10% FBS. Py2T cells, originally isolated from a breast tumor of a MMTV-PyMT female mouse with an FVB/N background, were a kind gift of Dr. G. Christofori (University of Basel, CH) were cultured in DMEM plus 10% FBS as described (Waldmeier et al., 2012).  NMuMg cells were maintained in DMEM supplemented with 2% and 1% FBS, respectively, for 8-16 h prior to the addition of 10 ng/ml human recombinant TGF-B1 purchased from R&amp;D Systems.    Cell transfections and adenoviral infections.    The adenoviral vectors pAdCMVnull (AdNull) and pAdKHSRP (full-length human  KHSRP cDNA cloned into a Adenoviral-Type 5 backbone) were purchased from Vector Biolabs (Eagleville, PA, USA).  NMuMg cells were transfected with Lipofectamine 2000 (ThermoFischer). Stable NMuMg transfectants were obtained using G418 (Sigma) selection (800 ug/ml). Mock and shKHSRP NMuMG cells were cultured in 2% FBS-containing medium for 16 h and, subsequently either maintained in the same medium for 48 h or treated with 10 ng/ml TGF-B in 2% FBS-containing medium for 48 h.  Stable KHSRP knock-down was obtained using previously described silencing sequences cloned in pSuper.Neo and pSuper.Puro (Oligoengine).    Total RNA was isolated using either the miRNeasy mini kit (Qiagen) or QIAzol Lysis Reagent (Qiagen) and retro-transcribed (50-100 ng) using either miScript reverse transcription kit (Qiagen) or Transcriptor Reverse Transcriptase (Roche) according to manufacturers’ instructions.  Small RNA library was generated from a customer sample using the Illumina Truseq™ Small RNA Preparation kit according to Illumina’s TruSeq™ Small RNA Sample Preparation Guide.
shKC2
San Raffaele Scientific Institute
shKC2