On targetplus smartpool

siRNA-lipofectamine complexes were created using Lipofectamine RNAiMAX (Thermo Fisher Scientific) as per the manufacturer’s instructions. All siRNAs used in this study were Dharmacon ON-TARGETplus SMARTpools (GE Healthcare). siRNA was added at a final concentration of 20 nM and allowed to grow for 24 hours. Cells were subsequently washed and fresh media applied to avoid lipofectamine toxicity. For knock-down confirmation, cells were trypsinised after 3 days in fresh media, collected and lysed in Cell Panel Lysis Buffer (5 mM Tris-HCl, 3 mM EDTA, 3 mM EGTA, 50 mM NaF, 2 mM sodium orthovanadate, 0.27 M sucrose, 10 mM ß-glycerophosphate, 5 mM sodium pyrophosphate, and 0.5% Triton X-100) supplemented with complete protease and phosSTOP phosphotase inhibitors (both Roche). Examples of knock-down efficiency are shown with the figures. These cells were subsequently used for down-stream experiments discussed below.

The chemicals quercetin (Q4951; Sigma-Aldrich, ON, Canada) and ICG-001 (S2662; Selleck chemical, TX, USA) were dissolved in DMSO and added to the cells 15h before co-treatment with TGFB1. Unless indicated otherwise, human recombinant TGFB1 (240-B; R&D Systems, MN, USA) was added to cells at a final concentration of 6.4 ng/ml for 48h.
The plasmids coding for ST8SIA2 (MR205823) and ST8SIA4 (MR205502) were purchased from Origene (MD, USA). Transient knockdowns were achieved with SilencerSelect siRNAs against Prnp (s72188; Life Technologies) and Ctnnb1 (s63418; Life Technologies). ON-TARGETplus SMARTpools were obtained from GE HealthCare (ON, Canada) to target St8sia2 (L-042781-01-0005) and St8sia4 (L-044724-01-0005) transcripts.
Immunoblotting made use of antibodies against NCAM1 (1:6666, 556324 or 556325; BD Biosciences, ON, Canada), PrP (1:2000, A03213; Bertin Pharma, France), E-cadherin (1:4000, 3195; Cell signaling, MA, USA), p-CREB (1:1000, 9198; Cell Signaling), SNAI1 (1:1000, 3879; Cell Signaling) and Lamin A (1:1000, 26300; Abcam). For the microscopy analyses, the Alexa Fluor 488 goat anti-rabbit IgG (1:200, A31627) and Alexa Fluor 647 phalloidin (1:200, A22287) antibodies were purchased from Life Technologies (ON, Canada).

Protein silencing was achieved using the Neon transfection system (Invitrogen, Life Technologies). Briefly, B cells were washed in phosphate-buffered saline (PBS), resuspended in Buffer R at a density of 50 × 10⁶ cells per ml and ON-TARGETplus SMARTpool Non-targeting (siCtrl), HS1- (siRNA#1: 5′-GGGCAUGAUGUAUCGGUUU-3′; siRNA#2: 5′-CCAAGGAGAGGGAAGCGAU-3′; siRNA#3: 5′-UGGAAGAGCCAGUGUACGA-3′; and siRNA#4: 5′-GUAAAGAUGAGCCGAGAAG-3′), Arp2- (siRNA#1: 5′-UGGUGUAACUGUUCGAUAA-3′; siRNA#2: 5′-GUUCUUUACUAAUGACGAA-3′; siRNA#3: 5′-GAUCAGUGCUUCUCGACAA-3′; and siRNA#4: 5′-CAUCGAGGUUGGAACGAGA), Arp3- (siRNA#1: 5′-GAAGAGAGCUAAGACGAUU-3′; siRNA#2: 5′-AAGCAGUGAAGGAACGCUA-3′; siRNA#3: 5′-GCUGACGGGUACAGUAAUA; siRNA#4: 5′-GAGUCAACGCCAUCUCAAA) or WASH- (siRNA#1: 5′-ACAGCAACACGGCGGAAUA-3′; siRNA#2: 5′-GAGGAGAAAUUGUUCGAUG-3′; siRNA#3: 5′-GCACAUUCAGGAACGUUUA-3′; and siRNA#4: 5′-GAAUACGGCUCCAUCUUUA-3′) targeting siRNA (Dharmacon, GE Healthcare) were added at a final concentration of 200 nM. Cells were then electroporated (1,300 V, 2 pulses, 20 ms per pulse) using 10-μl tips, and incubated in CLICK medium for 60–72 h. Silencing efficiency was analysed by western blot as described below.

siRNA targeting human USP11 or DDB1 (ON-TARGETplus SMARTpool) and Control siRNA (ON-TARGETplus Non-targeting siRNA) were purchased from GE Healthcare Dharmacon Inc. Nucleofector (Amaxa, Gaithersburg, MD) was used to electroporate cells with siRNA as previously described [57 (link), 60 (link), 61 (link)]. pRK5myc plasmids with wild-type (WT) and C275/283S mutant (csmt) USP11 were kindly provided by Dr. Ruey-Hwa Chen. The plasmids were transfected into HEK293T ΔUSP11 cells using X-tremeGENE 9 according to the manufacturer’s instructions (Roche) as described previously [62 ].