Doxycycline

The pTET/KRAS^G12V-IRES-GFP-bsr expression vector, which allows the doxycycline-inducible expression of K-Ras^G12V, was generated by recovering the K-Ras^G12V open reading frame from pBABE-K-Ras^G12V (Addgene) by BamHI digestion, followed by the insertion into the BglII-linearized pMIG vector [21] (link). The resulting K-Ras^G12V-IRES-GFP cassette was amplified by PCR using oligonucleotides containing flanking NotI sites, which were used for subcloning into pSC-A-amp/kan (Stratagene, La Jolla, CA, USA). Subsequently, the K-Ras^G12V-IRES-GFP cassette was cloned via NotI digestion into the pTET-bsr vector [22] , yielding pTET/K-Ras^G12V-IRES-GFP-bsr. To achieve doxycycline-inducible expression of oncogenic K-Ras, Caco-2tet cells, stably expressing the doxycycline-inducible system components rtTA and rtTS [21] (link), were transfected with the AhdI-linearized pTET/K-Ras^G12V-IRES-GFP-bsr vector by electroporation. Following selection with blasticidine S (5 µg/ml) and puromycin (5 µg/ml), resistant cell pools were screened for efficient induction of K-Ras^G12V expression. Transgene expression was induced by addition of 2 µg/ml doxycycline (Merck).

Primary epidermal progenitor cells were established from postnatal day (P) 0 pups and maintained according to published protocols (Nowak and Fuchs, 2009 (link)) using 3T3-J2 feeders and E-Low media [75% DMEM (Gibco, 21068028), 25% F12 (Gibco,21700026), supplemented with 15% chelated fetal bovine serum (FBS) (Hyclone, #SH30071.03), 0.45 µg/ml hydrocortisone (Merck, 386698-25MG), 0.1125 nM Cholera toxin (Sigma-Aldrich, D0564-1MG), 50 µg/mL transferrin (Sigma-Aldrich, T-2252), 50 µg/ml insulin (Sigma-Aldrich, I-5500), 0.02 nM 3T (3,3′,5-triiodo-L-thyronine, Sigma-Aldrich, T-2752), 1× penicillin-streptomycin (Gibco, 15140122), 8 mM L-glutamine (Gibco, 25030081) and 0.03% sodium bicarbonate (Gibco, 25080094)]. E-low media contained 63.53 µM calcium and in vitro differentiation media contained 1.5 mM calcium. Epidermal progenitor cells expressing shId1, shCebpa, shTcf3/4/12, control shScr or protein coding sequences for ID1 or CEBPA were generated by lentiviral infection followed by puromycin (1 µg/ml) selection. ID1 and CEBPA expression was induced by doxycycline (1 µg/ml) for 3 days using the pCW75.1 backbone (Addgene plasmid #38240). Primary epidermal progenitors were within 20 passages.

We cloned template DNA for the indicated guide RNAs into a pLX-sgRNA construct additionally containing blasticidin resistance (Addgene plasmid 50662). We transfected constructs into HEK293T cells along with lentivirus packaging vector pSPAX2 (Addgene plasmid 12260) and lentivirus envelope vector VSV-G (Addgene plasmid 8454). We used the resulting virus particles to transduce immortalized wild-type C57BL/6 cells that express doxycycline-inducible SpCas9 enzyme (generated using Addgene plasmid 50661). We cultured transduced cells in 3.0 μg/ml blasticidin (InvivoGen) and 5.0 μg/ml doxycycline (Sigma) for at least 2 weeks prior to use in experiments.

Doxycycline, puromycin, and crystal violet were purchased from Sigma-Aldrich (USA). Human PIERCE1 cDNA was subcloned into the following vectors: pCW57-RFP-P2A-MCS (Addgene, Cambridge, MA, USA) for Doxycycline inducible plasmid, pCS4-3×flag for Flag-tagging, pEGFP-C1 for GFP-tagging. pLNCX-myr-HA-AKT1 plasmid was purchased from Addgene (USA) and used to generate stable cell lines. Lentiviral vectors for shPIERCE1 expression were purchased from Sigma-Aldrich (USA). siRNAs for control, PIERCE1, RICTOR, RAPTOR, and TRIB3 were purchased from GenePharma. Lipofectamine 3000 (Invitrogen, USA), Lipofectamine RNAiMAX (Invitrogen, USA), and jetPEI (Polyplus-transfection SA, France) were used for transfection.

For generation of doxycycline inducible RAW MΦ, pLenti CMV rtTA3 Blast (Addgene, w756-1) stably expressing clonal RAW MΦ were transduced with pLenti CMV Puro DEST (Addgene, w118-1) constructs containing GFP-Strep, Bax201-Strep, Bax203-Strep, BaxG179P-Strep, GFP-FL, SRSF6-FL, and all SRSF6-FL phosphorylation mutants. After 48 hr, construct containing cells were selected through addition of puromycin (Invivogen, ant-pr-1). 1 mg/mL doxycycline (Calbiochem, 324385) treatment was used to activate construct expression.