Journal: Nature Communications
Article Title: Eukaryote initiation factor 6 modulates small-cell lung carcinoma plasticity via the integrin-FAK signaling axis
doi: 10.1038/s41467-026-69899-8
Figure Lengend Snippet: a Enrichment of EMT and NE gene signatures following EIF6 knockdown in non-NE cell states. RNA-seq analysis is performed in n = 2 biologically independent samples. b Gene expression profiling of EMT- and NE-associated pathways upon EIF6 knockdown in non-NE cell states. RNA-seq analysis is performed in n = 2 biologically independent samples. c Immunoblot analysis of EMT-associated and NE proteins following EIF6 knockdown across multiple SCLC models. Blots are representative of n = 3 biologically independent experiments. For H69M, the samples derive from the same experiment but different gels for ZEB1, another for ZEB2, another for MYCN, another for YAP1, another for eIF6 and c-Myc, another for ASCL1, NEUROD1 and β-tubulin were processed in parallel; For H82R, the samples derive from the same experiment but different gels for ZEB2, ZEB1, MYCN and c-Myc, another for YAP1 and ASCL1, another for NEUROD1, eIF6 and β-tubulin were processed in parallel. For H209A, the samples derive from the same experiment, but different gels for ZEB2, ZEB1, MYCN and c-Myc, another for YAP1 and ASCL1, another for eIF6, another for NEUROD1 and β-tubulin were processed in parallel. d H69 cells with EIF6 knockdown were treated with HGF (40 ng mL⁻¹) for two weeks. Bright-field images show adherent growth (top). Attached colonies were stained with crystal violet for quantification ( n = 3 biologically independent experiments). Scale bar, 100 μm. Error bars represent s.d. Statistical significance was assessed using a two-sided unpaired Wilcoxon rank-sum test. e Re-expression of shRNA-resistant wild-type EIF6 using pCDH lentivirus restores HGF-induced cell attachment in EIF6 knockdown cells. Top: immunoblot showing EIF6 knockdown and re-expression efficiency. Bottom, quantification of crystal violet–stained colonies ( n = 4 independent experiments). Error bars represent s.d. Statistical significance was assessed using a two-sided unpaired Wilcoxon rank-sum test. f Representative images of H69 cells with EIF6 knockdown and re-expression after HGF treatment (40 ng mL⁻¹) for three weeks. Bright-field images (left) and crystal violet–stained colonies (right) are shown. Scale bar, 20 μm. g Live/dead cell analysis of shNTC and shEIF6 H69M cells treated with or without EP (carboplatin 10 μM and etoposide 2 μM) for 72 h, measured by flow cytometry. Data are mean ± SEM from n = 3 independent experiments. Statistical significance was determined using a two-sided unpaired Wilcoxon rank-sum test. h Chemotherapy sensitivity of subcutaneous xenografts derived from shNTC or shEIF6 H69 cells. Tumour volume changes were assessed after three chemotherapy cycles ( n = 13 tumours per group). Response categories were defined as CR (−100%), PR (−30% to 0%) and PD (>20%). Lowercase letters indicate tumours used for immunohistochemistry in i . Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . i Representative immunohistochemistry images of ASCL1, NEUROD1, POU2F3, YAP1 and EIF6 in corresponding tumours from h . Scale bars, 50 μm.
Article Snippet: Lentiviruses were made by co-transfection of the shRNA lentivirus vector and the packaging plasmid psPAX2 (addgene#12260) and pMD2.G (addgene#12259) through calcium phosphate co-precipitation into 293 T cells.
Techniques: Knockdown, RNA Sequencing, Gene Expression, Western Blot, Staining, Expressing, shRNA, Cell Attachment Assay, Cell Analysis, Flow Cytometry, Derivative Assay, Immunohistochemistry
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