Control shrna lentivirus sc 108080

Lentiviruses used for knocking down GSK-3β and its control were obtained from Santa-Cruz (GSK-3β shRNA lentivirus: sc-270460-V; control shRNA lentivirus: sc-108080), and Adeno-associated viruses used for knocking in of phosphorylation-resistant form (S9A) of GSK-3β were generously provided by Dr. Yoon et al.^{85 (link)}. The viruses were produced by using AAV Helper-Free System (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's recommended protocol. Briefly, pAAV expression vector, pAAV-RC (DJ) and pHelper were transfected into AAV-293 cells. They were further incubated for 66–72 h. Then cells were harvested and went through repeated freeze/thaw cycles. The debris were centrifuged and the supernatant was mixed with 40% (vol/vol) PEG (to final 10% PEG) and left at 4 °C for 36–48 h. The viral particles were obtained from further centrifugation and resuspension in ice-cold PBS for two times. The GSK-3β S9A [MSGRPRTTAFAES…] containing plasmid was constructed from an original vector pAAV-ef1a-DIO-eYFP (from Karl Deisseroth, addgene #27,056, Water Town, MA, USA). The final form is pAAV-ef1a-S9A-P2A-eYFP where P2A is a linker peptide that is cleaved post-translationally, and the cleavage produces functional S9A and eYFP proteins. The viral titer (~ 1.0 × 10⁸ infectious unit/µl) was measured in HT-1080 cells via flow cytometry.

Recombinant TGFβ1 was obtained from R&D Systems (Minneapolis, MN); normal rabbit IgG, normal mouse IgG, and ChIP-Grade Protein A/G Magnetic Beads were obtained from Thermo Scientific, (Rockford, IL, USA). The following antibodies were used in western-blot analyses and ChIP assays: anti-smad3 (ab28379), anti-SET7/9 (ab13731), anti-histone H3 (ab100938), anti-histone H3 trimethyl K9 (ab8898), and anti-H3 trimethyl K27 (ab1782) from Abcam (Cambridge, MA); anti-MLL1 (05–765), anti-H3 monomethyl K4 (07–436), and anti-H3 trimethyl K4 (07–473) from Millipore (Millipore Corp., Bedford, MA); anti-TGFBIp (AF2935) from R&D Systems; and anti-β-actin (A-5441) from Sigma-Aldrich (St. Louis, MO). MLL1 shRNA lentivirus (sc-38039-v), SET7/9 shRNA lentivirus (sc-44094-v), and control shRNA lentivirus (sc-108080) was purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Lentivirus containing small hairpin RNA (shRNA) targeting human USP14 (sc-76817-V) and control shRNA lentivirus (sc-108080) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were cultured in 24-well cell culture plate at density of 2 × 10⁴ cells per well and infected with control shRNA lentivirus or USP14 shRNA lentivirus according to the manufacturer’s protocol. Control (Ctrl) THP-1 cells, USP14-knockdown (USP14^KD) THP-1 cells, Ctrl MDA-MB-231 cells, USP14^KD MDA-MB-231 cells, Ctrl SK-HEP-1 cells, and USP14^KD SK-HEP-1 cells were cultured in puromycin-containing (4–8 µg/ml) medium for 2 weeks to select stable clones and cultured as described previously (25 (link), 26 (link)).