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Figure 4. Hsa-circ-0001030 directly binds to PKM2 and inhibits its glycolytic function in TSCC cells. (A) RNA pulldown assays were performed using biotin-labeled specific and non-specific probes for hsa-circ-0001030, and the retrieved proteins were separated by SDS–PAGE; (B and C) Marked enrichment of PKM2 in Cal27 and Cal27R cell lysates pulled down with the hsa-circ-0001030–specific probe was shown by Western blot analysis; (D and E) RIP assays confirming the interaction between PKM2 and hsa-circ-0001030 in Cal27 and Cal27R cells using anti-PKM2 antibody compared with IgG control; (F) Predicted secondary structure of hsa-circ-0001030 based on MFE was generated by the RNAfold WebServer; (G and H) Segmented RNA pulldown analysis identifying the 138-169 nt region of hsa-circ-0001030 as the binding site for PKM2; (I and J) Western blot detection of PKM2 phosphorylation status showing that hsa-circ-0001030 overexpression increases Tyr105 phosphorylation without affecting Ser37 phosphorylation; (K and L) Immunofluorescence analysis showing that hsa-circ-0001030 overexpression prevents PKM2 nuclear translocation, with PKM2 predominantly localized in the cytoplasm; (M and N) MTT assays assessing cisplatin IC₅₀ values in hsa-circ-0001030–overexpressing Cal27R and Tca8113R cells treated with the PKM2 agonist TEPP-46, showing enhanced cisplatin sensitivity; (O and P) Representative Transwell images demonstrating that hsa-circ-0001030 overexpression suppresses migration and invasion of Cal27R and Tca8113R cells, whereas treatment with the PKM2 agonist TEPP-46 partially reverses these effects. Data are presented as mean ± SD from three independent experiments. PKM2: Pyruvate kinase M2; TSCC: tongue squamous cell carcinoma; SDS–PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; RIP: RNA-binding protein immunoprecipitation; MFE: minimum free energy; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC₅₀: half-maximal inhibitory concentration; SD: standard deviation; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6-diamidino-2-phenylindole.