Journal: bioRxiv
Article Title: Dual-compartment engagement of STAR-family proteins SAM68 and QKI by LINC00941 sustains oncogenic fitness in RAS-driven lung cancer
doi: 10.64898/2026.05.11.722569
Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of LINC00941 expression in A549, H358, H1299, and H1437 cells following transfection with non-targeting siRNA (NT), non-targeting antisense oligonucleotide (AN), siLINC00941 (S1, S2,S3 and S4), or ASO-LINC00941 (A1, A2), confirming efficient knockdown. (B) Cell proliferation assays in A549, H358, H1299, and H1437 cells following transfection with siNT or siLINC00941. Proliferation was significantly reduced in A549, H358, and H1299 cells, whereas H1437 cells, which lack LINC00941 expression, showed no significant change. (C) Long-term colony formation assay in A549, H358, and H1299 cells treated with siNT, siL- INC00941, ASO-NT, or ASO-LINC00941. Cells were fixed and stained with crystal violet after two weeks. Representative images and quantification show a significant reduction in colony formation upon LINC00941 knockdown. (D) Cell proliferation analysis in A549, H358, and H1299 cells treated with ASO-NT or ASO- LINC00941, demonstrating a significant decrease in proliferation following LINC00941 depletion. (E) In vivo tumour growth assay in which H1299 cells stably expressing control shRNA or shLINC00941 were injected subcutaneously into NOD/SCID mice. Tumour volume was measured at the indicated time points, showing significantly reduced tumour growth upon LINC00941 knockdown. (F) BrdU incorporation assay in A549, H358, and H1299 cells following treatment with siNT, siL- INC00941, ASO-NT, or ASO-LINC00941, indicating reduced DNA synthesis upon LINC00941 depletion. (G) Caspase-3/7 activity measured using a Promega luminescence assay in H1299 cells treated with siNT, siLINC00941, ASO-NT, or ASO-LINC00941. No significant increase in apoptotic activity was observed following LINC00941 knockdown. (H) Senescence-associated β -galactosidase staining in A549, H1299, and H358 cells transfected with siNT or siLINC00941. LINC00941 depletion resulted in a significant increase in β -galactosidase-positive cells compared with controls. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test unless otherwise indicated. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
Article Snippet: LentiX cells were co-transfected with the shRNA expression vector and packaging plasmids using X-fect transfection reagent (Takara Bio, Japan Bio, Japan) according to the manufacturer’s instructions.
Techniques: Quantitative RT-PCR, Expressing, Transfection, Knockdown, Colony Assay, Staining, In Vivo, Growth Assay, Stable Transfection, Control, shRNA, Injection, BrdU Incorporation Assay, DNA Synthesis, Activity Assay, Luminescence Assay
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![(A) Heat map of differentially expressed genes identified by RNA-seq analysis comparing LINC00941 knockdown and control cells. Heat map showing expression of the same set of differentially expressed genes obtained from microarray analysis comparing siLINC00941 and siNT conditions using two independent siRNAs. (B) Volcano plot showing differentially expressed genes following LINC00941 knockdown in H1299 cells. Cells were transfected with two independent siRNAs targeting LINC00941 or four non-targeting siRNAs (siNT) for 48 h prior to RNA isolation and RNA sequencing. (c) KEGG pathway enrichment analysis of differentially expressed genes upon LINC00941 knock-down. Bar height indicates enrichment score and colour represents − log 10 ( P value). (D) Pre-ranked gene set enrichment analysis (GSEA) using Hallmark gene sets based on RNA-seq data. DNA replication and double-strand break repair pathways were among the most significantly negatively enriched gene sets following LINC00941 depletion. (E) Comet assay performed in control and LINC00941-depleted H1299 cells 48 h post-transfection. Representative images are shown and tail length was quantified using Comet Analyser software. (F) Immunoblot analysis of DNA damage response signalling proteins in H1299 cells treated with siNT, siLINC00941, ASO-NT, or ASO-LINC00941, demonstrating reduced activation of DDR pathways upon LINC00941 knockdown. (G) Immunofluorescence staining of γ H2AX in control and siLINC00941-treated H1299 cells. Representative images are shown and γ H2AX foci were quantified, revealing a significant reduction in foci number following LINC00941 depletion. (H) RNA fluorescence in situ hybridisation (RNA-FISH) analysis of LINC00941 localisation in H1299 cells. Probes targeting LINC00941 exons were labelled with Cy3 (green), and intronic probes were labelled with Cy5 (red). Nuclei were counterstained with DAPI. LINC00941 signal was detected in both the nuclear and cytoplasmic compartments as evident from the merged image. (I) Subcellular fractionation of H1299 cells followed by quantitative RT-PCR analysis of LINC00941 expression in nuclear and cytoplasmic fractions. H19 and HOTAIR were used as control lncRNAs for cytoplasmic and nuclear localisation, respectively. (J) RNA stability assay in H1299 cells treated with the transcriptional inhibitor actinomycin D. RNA was isolated at the indicated time points and LINC00941 expression was measured by quantitative RT-PCR and normalised to actin mRNA. (K) Schematic overview of the experimental workflow used to identify LINC00941-interacting proteins in whole-cell, nuclear, and cytoplasmic fractions. In vitro -transcribed, biotin-labelled LINC00941 RNA was incubated with cell lysates, followed by streptavidin pulldown and mass spectrometry analysis. (L) Table listing proteins enriched in LINC00941 pulldown samples from whole-cell, nuclear, and cytoplasmic lysates as identified by mass spectrometry. (M) Venn diagram showing overlap of LINC00941-associated proteins across nuclear and cyto-plasmic fractions. KHDRBS1 (SAM68) was uniquely enriched in the nuclear fraction, whereas QKI was enriched in the cytoplasmic fraction. (N) Bar graph depicting spectral counts from RNA immunoprecipitation (RIP), showing FAM120A binding to LINC00941 and [Control RNA] across nuclear, cytoplasmic, and whole-cell fractions. FAM120A demonstrated robust association with LINC00941 in all three fractions, while binding to [Control RNA] was negligible, confirming the specificity of the FAM120A–LINC00941 interaction. (O) Violin plot illustrating the differential expression of FAM120A protein in patient samples stratified by LINC00941 expression levels (high versus low). FAM120A expression was significantly elevated in LINC00941-high samples compared to LINC00941-low samples, indicating a positive correlation between FAM120A abundance and LINC00941 expression in clinical specimens. (P) Bar graph showing LINC00941 RNA and FAM120A RNA levels upon FAM120A knockdown relative to control cells. FAM120A depletion resulted in a significant reduction in LINC00941 RNA abundance, suggesting that FAM120A positively regulates LINC00941 expression or stability. (Q) RT-qPCR analysis of LINC00941 RNA expression in nuclear and cytoplasmic fractions of H1299 cells following FAM120A knockdown. RNA levels are presented relative to control. (R) Actinomycin D chase assay in control and FAM120A-depleted H1299 cells. Transcription was blocked with actinomycin D and LINC00941 RNA levels were measured by RT-qPCR at 0, 4, 8, and 12 hours. Values are normalised to the 0 h timepoint and expressed as percentage RNA remaining. Exponential decay fitting yielded half-lives of 25.7 h (control) and 5.1 h (FAM120A KD). Data are presented as mean ± SEM unless otherwise indicated. Statistical significance was determined using Student’s t -test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_69/10__64898_slash_2026__05__11__722569/10__64898_slash_2026__05__11__722569___F3.large.jpg)