L 011690 01 0005

In knockdown experiments using WT or TARDBP KO HeLa cells, cells were incubated with siLentfect (Bio-Rad) and siRNA complexes: AllStars Neg. Control siRNA (QIAGEN, control for TDP-43 knockdown assay, Cat# 1027281), siGENOME Control Pool Non-Targeting (Dharmacon, control for other hnRNPs knockdown assay, Cat# D-001206-13-20), siRNA against TARDBP 3′ UTR (DNA target sequence: 5′-AAGAGTTGTCATTGTTGGAAA, QIAGEN), or siRNA against HNRNPL (L-011293-01-0005, Dharmacon), HNRNPA1 (L-008221-00-0005, Dharmacon) or HNRNPA2B1 (L-011690-01-0005, Dharmacon) following manufacturer’s instructions for 48 h. All experiments were replicated 3 or 4 times.

Two micrograms of plasmid expressing control or AUF1 shRNAs (pSilencer-U6-hygro/shCTRL or pSilencer-U6-hygro/shAUF1) [22] (link) were mixed in 100 µl serum-free MEM and combined with 10 µl SuperFect reagent (Qiagen); this mixture was incubated at room temperature for 10 min before transfecting into cells. To reduce expression of hnRNP A1 and hnRNP A2, SF268 cells were seeded in 12-well plates in antibiotic-free media. One hundred nmol of each siRNA targeting hnRNP A1 and hnRNP A2 (ON-TARGETplus SMARTpool L-008221-00-0005 or L-011690-01-0005, Dharmacon) was transfected along with 3 µl Lipofectamine 2000 transfection reagent (Life Technologies) in 0.4 ml RPMI supplemented with 10% FCS following the manufacturer’s directions. To co-transfect shAUF1 and siRNAs targeting hnRNPs A1 and A2, Lipofectamine 2000 was used. Knockdown efficiency was monitored by Western blotting.