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Figure 4. LINC01607 depletion increases ferroptosis-associated vulnerability in HCC. (A) PCA plot of RNA-seq profiles from siLINC01607- and siNC-transfected Hep3B cells. The PCA shows distinct clustering between the control and LINC01607-knockdown groups, indicating significant transcriptional changes; (B) GO and KEGG enrichment analyses were performed on differentially expressed genes and highlighted antioxidant-related functions and ferroptosis-associated pathways; (C) Cell viability assays (CCK-8) in Hep3B cells treated with ferrostatin-1 (20 μM), liproxstatin-1 (2 μM), Z-VAD-FMK (20 μM), necrostatin-1 (20 μM), or Baf-A1 (100 μM) for 24 h, followed by cotreatment with the ferroptosis inducer erastin (5 μM) for 48 h. Ferrostatin-1 and liproxstatin-1 rescued the proliferation-suppressive effects of LINC01607 knockdown, while the other inhibitors did not; (D) Intracellular Fe²⁺ levels were measured by colorimetric assay and were increased in LINC01607-silenced cells treated with erastin; (E) GSH levels were reduced after LINC01607 knockdown compared with controls; (F) Flow cytometry was used to assess intracellular lipid ROS accumulation under the indicated treatment conditions; (G) Representative TEM images of Hep3B cells demonstrated ferroptosis-associated mitochondrial shrinkage and increased membrane density in LINC01607-knockdown cells; (H) Immunofluorescence images illustrating red-to-green fluorescence shifts indicative of lipid ROS accumulation under the indicated treatments by C11 staining; (I) Western blot analysis of ferroptosis-related proteins, including SQSTM1, NRF2, NQO1, HO-1, GPX4, and SLC7A11, revealed decreased expression in LINC01607-knockdown cells and increased expression in LINC01607-overexpressing cells; (J) Ferrostatin-1 partially restored cell proliferation in LINC01607-knockdown HCC cells, as assessed by CCK-8 assays; (K) Ferrostatin-1 also attenuated the inhibitory effects of LINC01607 knockdown on migration and invasion in Transwell assays, with quantification shown. Data are presented as mean ± SD for quantitative assays. RNA-seq analysis included three independent biological replicates per group. For cell viability, intracellular Fe²⁺, GSH, lipid ROS, and rescue assays, n = 3 independent biological replicates unless otherwise indicated. Statistical significance was assessed using two-tailed Student’s t-test for two-group comparisons and one-way ANOVA for multiple-group comparisons, as appropriate. ^**P < 0.01, ^***P < 0.001; ns, not significant. HCC: Hepatocellular carcinoma; PCA: principal component analysis; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; CCK-8: Cell Counting Kit-8; GSH: glutathione; ROS: reactive oxygen species; TEM: transmission electron microscopy; SD: standard deviation; ANOVA: analysis of variance; TNF: tumor necrosis factor; FITC: fluorescein isothiocyanate.