Anti hnrnp 1

Immunohistochemical analysis for the protein expression was performed as previously described [26] using anti-hnRNP I (1:500; Santa Cruz Biotechnology, Inc; CA, USA) anti-hnRNP L (1:500; Santa Cruz Biotechnology, Inc; CA, USA ) GPT2 (1:300; Abcam, Cambridge, MA, USA ). Slides were incubated overnight at 4 °C, and immunostaining was performed with a SPlink Detection Kit and DAB (ZSBIO, Beijing, China). Stained slides were captured and imaged by microscopy. The image quanti cation was analyzed using ImageJ(1.52q) with the IHC Pro ler plugin [27] . The staining intensity was graded as negative (0), low positive (1+), positive (2+), or high positive (3+).
RNA pull-down RNA pull-down assays were performed using PierceTM Magnetic RNA-Protein Pull-Down Kit (#20164, Thermo Fisher Scienti c, Waltham, MA, USA) according to the manufacture's protocol. The RNA fragments of the entire UCA1 sequence, deleted form, or mutant form, were synthesized by GenePharma (Shanghai, China).
Biotinylated RNA was bound to streptavidin magnetic beads and then incubated with cell lysates. RNA-binding protein complexes were washed and eluted. The retrieved samples were heated (10 minutes, 95℃) and detected by western blot analysis.

Block NC membrane was incubated overnight at 4°C with different primary antibodies: anti-hnRNP I (1:1000; Santa Cruz Biotechnology, Inc; CA, USA); anti-hnRNP L (1:1000; Santa Cruz Biotechnology, Inc; CA, USA); anti-GLS2 (1:1000); anti-GPT2 (1:1000; Abbacy, Cambridge, UK); anti-SLC1A5(1:500); anti-SLC7A5(1:500); anti-β actin (1:3000; CST; Beverly, MA, USA). After being washed, the membranes were incubated with HRPconjugated goat-anti-mouse secondary antibodies (1:5000; Pierce; Rockford, IL, USA) for 1 hr at room temperature and visualized using Clarity™ Western ECL Substrate (Bio-Rad; Richmond, CA, USA).

ChIP experiments were performed using EZ-Magna ChIP TM A/G Chromatin Immunoprecipitation Kit (#17-10086, Merck Millipore, Billerica, MA, USA) according to the manufacturer's procedures. Cells were xed in 1% formaldehyde for 10 min at room temperature to crosslink UCA1 to DNA. Nuclei were isolated with 500 μl nuclear lysis buffer supplemented with 2.5 μl protease inhibitor cocktail II. Chromatin DNA was sonicated (8min total, AmpL 30%, pulse on 10 s, pulse off 30 s) and sheared to a length between 200 bp to 1000 bp. The sheared cross-linked chromatin was incubated and rotated with immunoprecipitating antibodies: anti-hnRNP I (1.2μg/reaction; Santa Cruz Biotechnology, Inc; CA, USA)), anti-hnRNP L (1μg/reaction; Santa Cruz Biotechnology, Inc; CA, USA), normal Mouse IgG (1μg/reaction) or anti-RNA Polymerase (1μg/reaction) overnight at 4 °C. Primers for ChIP-qPCR are listed in Table 2.