D 001206 13 20

NCCIT and HCT116 parental cells (from ATCC), and the isogenic derivatives of parental HCT116 (Rhee et al., 2002 (link); Sun et al., 2008 (link)) were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum and Lglutamine. HCT116 DNMT overexpression lines (Sun et al., 2008 (link)) were grown under G418 selection (750 μg/ml). Differentiation of NCCIT was induced by addition of 10 μM all-trans retinoic acid (Sigma) for 7 days. On-TARGETplus and siGENOME siRNA SMARTpools (Dharmacon, Thermo Scientific) targeting a single gene were used against DNMT1 (L-004605-00-0005), DNMT3A (M-006672-03-0005), DNMT3B (L-006395-00-0005), and DNMT3L (L-013637-01-0005) in individual and combination experiments. siRNA transfection with a negative control non-targeting siRNA (D-001206-13-20; Dharmacon, Thermo Scientific) was performed in parallel. siRNA transfection was performed with PepMute transfection reagent (SignaGen) according to the manufacturer protocol. Total RNA was extracted by Trizol homogenization and purified according to the manufacturer protocol (Life Technologies). Genomic DNA was extracted by proteinase K digestion and phenol:chloroform extraction. Refer to Supplemental Experimental Procedures for the full siRNA transfection protocol and siRNA rescue experimental conditions.

We used VIM specific Accel smart pool siRNA to knock
down vimentin in PC3 cells, according to the manufacturer’s protocol.
Briefly, PC3 (100,000 cells) were cultured at 37°C and 5% CO2 in 6-well
plate overnight. A stock solution of 100 μM VIM specific
Accel smart pool siRNA (E-0003551-00-0020, Dharmacon, US) in 1x siRNA buffer
(B-002000-UB-100) was prepared. The siRNA was suspended in 300 μL Accel
delivery medium (B-005000-500, Dharmacon, US) to a final concentration of 1
μM and the suspension was shaken for 90 min at room temperature. The
suspension was centrifuged briefly. The culture medium was aspired from each
well and the cells were incubated with 300 μL siRNA suspension in the
Accel delivery medium for 96 h at 37°C and 5% CO2. A control experiment
was carried out in which the cells were incubated with 1 μM non-targeting
siRNA (D-001206-13-20, Dharmacon, US) in the Accel Delivery medium (300
μL) under the same conditions. Finally, the cells were trypsinized and
the vimentin protein level was analysed using the microfluidic approach and flow
cytometry.