Fig. 3: DARS-AS1 directly interacts with PACT.

a RNA pull-down assay identifies DARS-AS1 interacting with PACT in SW620 cells. Upper, silver staining of associated proteins. Lower, immunoblot with anti-PACT antibodies. b RNA pull-down assays were performed in HCT116 (upper) and HepG2 (lower) cells. Immunoblot detected the enrichment of PACT. c RNA immunoprecipitation (RIP) assays were performed using the indicated antibodies in SW620 cells. d The binding curves of PACT with full length DARS-AS1 or control RNA were obtained by biolayer interferometry (BLI). RNAs were immobilized on streptavidin biosensors. 1 μM PACT were used for measuring the association. e RNA pull-down assays were performed using biotinylated full length DARS-AS1 or truncations (upper). Immunoblot shows the retrieved PACT (lower). f Purified flag-tagged PACT was incubated with biotinylated full length DARS-AS1 or truncations (as showed in e) for in vitro RIP assays. Retrieved RNAs were validated by RT-qPCR. g The relative affinities of different RNA fragments for PACT were obtained by biolayer interferometry. 100 nM RNA and 1 μM PACT were used in each assay. h In vitro RIP assays were performed using purified intact or truncated flag-tagged PACT. Retrieved RNA was validated by RT-qPCR. i Cell growth rate of SW620 cells overexpressing DARS-AS1, PACT, or both. j Overexpression of full length or truncated DARS-AS1 in SW620 cells shows different impacts on cell growth. k Cell apoptosis was detected by immunoblot with anti-PARP antibodies. l Knockdown DARS-AS1 induces SW620 cell apoptosis, as revealed by flow cytometry. Data shown are means ± SD in triplicates experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, by two-tailed Student’s t test.