Fig 2: Density gradient sedimentation analysis showing that ILF2 is a component of HMM complexes. Whole cell lysates from AMO1 KI and RPMI8226 KI or lysis buffer containing albumin were loaded on to a discontinuous OptiPrep gradient (4 36%) and centrifuged at 25,000 rpm for 14 h at 4 °C. The separated fractions were analysed by Western blotting with specific antibodies against FLAG and each interactor candidate (left, AMO1, right, RPMI8226). The position of albumin as a molecular weight marker was confirmed using silver staining. LMM Low molecular mass. HMM High molecular mass. Original blots are presented in Supplementary Figure S14.

Fig 3: A3B co localizes with ILF2 and SAFB in the nucleus. AMO1 KI cells were subjected to immunofluorescence using Alexa Fluor 555 labelled anti mouse secondary antibody. Endogenous ILF2 (a) and SAFB (b) were visualized using Alexa Fluor 647 labelled anti rabbit secondary antibody and DAPI was used to stain the nucleus. Image acquisition was performed with a confocal laser scanning microscope (BZ X800, KEYENCE). WT, Wild type (parental) cell lines. KI, 3 × FLAG IRES EGFP knock in cell lines. HPF High power field.

Fig 4: LINC00571 interacts with HNRNPK protein and ILF2 protein. A Silver staining unveiled proteins interacting with LINC00571, with biotin-labeled sense or antisense LINC00571 probes used for RNA-protein pull-down against MDA-MB-231 cell lysates. B A simplified flowchart outlined the systematic screening process used to identify proteins that interacted with LINC00571. C Mass spectrometry analysis revealed HNRNPK peptides and ILF2 peptides pulled down by LINC00571 sense probes. D Immunoblot analyses were performed for HNRNPK and ILF2 on biotin-labeled sense and antisense LINC00571 probe pull-down eluates from MDA-MB-231 and BT-549 cell lysates, with GAPDH as a loading control. E RNA immunoprecipitation (RIP) was conducted on MDA-MB-231 and BT-549 cells using HNRNPK and IgG antibody or ILF2 and IgG antibody. The precipitates underwent immunoblot analysis with HNRNPK and GAPDH antibody or ILF2 and GAPDH antibody. HNRNPK or ILF2 enrichment of LINC00571 relative to IgG enrichment values was quantified by qRT-PCR. F RNA-FISH and immunofluorescence staining assays revealed subcellular co-localization of LINC00571 (green), ILF2 (red), and HNRNPK (cyan), along with nuclear staining using DAPI (blue). scale bar: 10μm (G) Schematic representation of HNRNPK with functional protein domains. HNRNPK had been truncated within regions: 1-143aa, 1-213aa, 144-213aa, 214-463aa, and 144-463aa. H-I Relative enrichment of endogenous LINC00571 in truncated HNRNPK RIP was measured by qRT-PCR, following MDA-MB-231 cells transfected with 3xFlag-HNRNPK truncations. Statistical analyses are depicted in bar graphs. Data are presented as mean ± SD from three independent experiments. Significance levels are denoted as * for p<0.05, ** for p<0.01, and *** for p<0.001, as determined by the t-test

Fig 5: The addition of α-KG rescues the phenotypes induced by ILF2 knockdown. (A-C) Proliferation rate status of MDA-MB-231 cells was assessed through CCK-8, colony formation assays, and EdU assays, n = 3. scale bar: 50μm. D Cell cycle analysis was conducted via flow cytometry, involving propidium iodide (PI) staining on MDA-MB-231 cells, n = 3. E Apoptosis detection was performed using a flow cytometry assay. Annexin V and propidium iodide (PI) staining were employed on MDA-MB-231 cells, n = 3. F Left, oxygen consumption rate (OCR) on addition of oligomycin (Oligo), fluorocarbonyl cyanide phenylhydrazone (FCCP) and rotenone plus antimycin A (R&A) (n = 4). Right, basal respiration, ATP-coupled respiration and maximal respiration (n= 4). G-H Relative lactate level (G) and relative ATP level (H) in MDA-MB-231 cells with ILF2 knockdown and α-KG supplementation were shown, n = 3. (I) Relative lactate level (left) and relative ATP level (right) in MDA-MB-231 cells with ILF2 knockdown and α-KG supplementation were shown, n = 3. J-N In vivo studies involved BALB/c athymic nude mice subcutaneously injected with MDA-MB-231 cells, n = 5. Images of xenograft s were captured using a digital camera (J). Tumor weight was measured on day 50 (K). Tumor volume was measured at ten-day intervals, calculated as volume = length × (width)2/2 (L). Immunohistochemical images showing Ki67 and PCNA staining in control, shILF2-, α-KG-, and shILF2 combined plus α-KG-treated groups. scale bar: 100μm (M). Immunofluorescence images showcased TUNEL staining in control, shILF2-, α-KG-, and shILF2 combined plus α-KG-treated groups. scale bar: 60μm (N). Statistical analyses are depicted in bar graphs. Data are presented as mean ± SD. Significance levels are denoted as * for p<0.05, ** for p<0.01, and *** for p<0.001, as determined by the t-test