Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A) Representative Transwell images of invasion (top) and migration (bottom) of U251 cells after LNA-mediated knockdown (KD) of tRF-3009a, tRF-3021a, or tRF-3030a compared with negative-control (NC) LNA. (B–C) Quantification of invasion (B) and migration (C) in U251 cells following tRF-3 KD. Cell numbers are normalized to NC LNA (set to 100%). (D) Representative Transwell images of invasion (top) and migration (bottom) of U251 cells after overexpression (OE) of tRF-3009a, tRF-3021a, or tRF-3030a using synthetic mimics compared with NC mimic. (E–F) Quantification of invasion (E) and migration (F) following tRF-3 OE. Cell numbers are normalized to NC mimic (set to 100%). (G) qRT–PCR analysis of EMT-associated transcripts ( CDH2, SNAI2, VIM, TWIST1 ) in U251 cells after tRF-3021a KD versus NC LNA; expression is normalized to GAPDH and shown relative to NC LNA (set to 1). (H) Immunoblot analysis of N-cadherin, SLUG, and TWIST1 in U251 cells after tRF-3021a KD versus NC LNA; GAPDH serves as a loading control (representative of n = 3 independent experiments). (I) U251 proliferation measured by MTT at 24, 48, and 72 h following KD of tRF-3009a, tRF-3021a, or tRF-3030a versus NC LNA. (J–K) MTT assays in normal astrocytes (J) and U87 glioma cells (K) after tRF-3021a KD versus NC LNA at the indicated time points. Data presentation: For quantified panels, data are mean ± SD from n = 3 independent experiments (B–C, E–F, I–K) or n = 3 biological replicates (G). Statistics: (B–C, E–F) one-way ANOVA with Dunnett’s multiple-comparisons test versus the corresponding NC. (G) unpaired two-tailed t tests with Benjamini–Krieger–Yekutieli false-discovery-rate correction (Q = 1%). (I–K) two-way ANOVA (time × treatment) with Sidak’s multiple-comparisons test comparing tRF-KD to NC LNA at each time point. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: 8 h: Total RNA from knockdown (KD) and negative control (NC) cells was submitted to Plasmidsaurus for 3’-RNA-seq.

Techniques: Migration, Knockdown, Negative Control, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A–B) Representative propidium iodide (PI) DNA-content histograms for cell-cycle profiling of U251 cells 48 h after transfection with negative-control (NC) LNA (A) or tRF-3021a LNA (B). (C-D) Quantification of sub-G1 population (C) and cell cycle distribution after subtracting the sub-G1 cells (D) from three independent experiments (n = 3). (E-F) Representative Annexin V/PI flow-cytometry plots of U251 cells 48 h after transfection with NC LNA (E) or tRF-3021a LNA (F). Quadrants indicate live (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic/necrotic (Annexin V⁺/PI⁺), and necrotic PI + only populations. (G) Quantification of Annexin V–positive cells (early + late apoptotic) in U251 cells after knockdown of tRF-3009a, tRF-3021a, or tRF-3030a versus the corresponding NC LNA. (H) Annexin V–positive cells (early + late apoptotic) after tRF-3021a knockdown versus NC LNA across astrocytes and the indicated tumor cell lines (U87, U2OS, HCT116, MCF7). Data presentation: Data are mean ± SD from n = 3 independent experiments for panels (C), (D) and (G). Statistics: (C) Unpaired two-tailed t test comparing tRF-3021a KD to NC LNA. (D) multiple unpaired two-tailed t tests with Benjamini–Krieger–Yekutieli false-discovery-rate correction (Q = 1%). (G) unpaired two-tailed t test comparing tRF-3021a KD to NC LNA. Panel (H) was performed once (no statistics). Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: 8 h: Total RNA from knockdown (KD) and negative control (NC) cells was submitted to Plasmidsaurus for 3’-RNA-seq.

Techniques: Transfection, Negative Control, Flow Cytometry, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A) Puromycin incorporation assays in the indicated cell lines following LNA-mediated knockdown (KD) of tRF-3021a versus negative-control (NC) LNA. Cells were incubated with puromycin, and lysates were immunoblotted with anti-puromycin to assess global protein synthesis; GAPDH serves as a loading control. (B) Puromycin incorporation in U251 cells after KD of tRF-3009a, tRF-3021a, or tRF-3030a compared with the corresponding NC LNA; GAPDH serves as a loading control. (C) Immunoblot analysis of translation-regulatory signaling in U251 cells 48 h after tRF-3021a KD versus NC LNA, including total and phospho-eIF2α, total and phospho-p70S6 kinase (Thr389), and total and phospho-4E-BP1 (Thr37/46); GAPDH serves as a loading control.

Article Snippet: 8 h: Total RNA from knockdown (KD) and negative control (NC) cells was submitted to Plasmidsaurus for 3’-RNA-seq.

Techniques: Knockdown, Negative Control, Incubation, Control, Western Blot

Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A) Time course quantification of apoptotic (Annexin V–positive) U251 cells after transfection with negative control (NC) LNA or tRF-3021a LNA at the indicated time points. (B) Immunoblot time course of cleaved PARP in U251 cells following tRF-3021a knockdown (KD) versus NC LNA; GAPDH serves as a loading control. (C) Puromycin -labeling time course in U251 cells following tRF-3021a KD versus NC LNA. Cells were incubated with puromycin, and lysates were immunoblotted with anti-puromycin to assess global protein synthesis; GAPDH serves as a loading control. (D) Time course immunoblot analysis (2–12 h) of translation-regulatory signaling following tRF-3021a KD versus NC LNA, including total and phospho-eIF2α, total and phospho-p70S6 kinase (Thr389), and phospho-4E-BP1 (Thr37/46); GAPDH serves as a loading control. (E) Quantification of apoptotic (Annexin V–positive) cells after tRF-3021a KD in the presence or absence of the pan-caspase inhibitor z-VAD-fmk. (F) Puromycin-labeling assay at 48 h in U251 cells transfected with NC LNA, tRF-3021a LNA, tRF-3021a LNA + z-VAD-fmk or treated with z-VAD-fmk alone. Statistics: multiple unpaired two-tailed t tests comparing KD to NC at each time point with Benjamini–Krieger–Yekutieli false-discovery-rate correction (Q = 1%); p = 0.007751 (12 h), p = 0.000205 (24 h), p < 0.000001 (48 h). (E) one-way ANOVA with Tukey’s multiple-comparisons test; p = 0.0316 for KD versus KD + z-VAD. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: 8 h: Total RNA from knockdown (KD) and negative control (NC) cells was submitted to Plasmidsaurus for 3’-RNA-seq.

Techniques: Transfection, Negative Control, Western Blot, Knockdown, Control, Labeling, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A, B) Volcano plot illustrating differential gene expression analysis at 8 h (A) and 48 h (B) after tRF-3021a knockdown in U251 cells. The x-axis represents the log-transformed fold change (logFC), and the y-axis represents the negative log-transformed false discovery rate (FDR). Each point represents a single gene. The vertical dashed lines indicate the logFC threshold (±1.0), and the horizontal dashed line indicates the significance threshold (FDR = 0.05). Genes are color-coded based on their regulatory status: orange represents significantly upregulated genes (logFC > 1.0, FDR < 0.05), purple represents significantly downregulated genes (logFC < −1.0, FDR < 0.05), and gray represents non-significant genes. The top significantly dysregulated genes are labeled with their gene symbols. (C) Overlap of significantly upregulated genes (FDR < 0.05, logFC > 1). (D) Overlap of significantly downregulated genes (FDR < 0.05, logFC < 1). The number of unique and shared genes in each comparison set is shown. (C,D) Venn diagrams showing the overlap of differentially expressed genes (DEGs) identified at 8 hand 48 h. (E, F) Gene Set Enrichment Analysis (GSEA) of MSigDB Hallmark pathways at 8 h (E) and 48 h (F). A bar graph showing the top 20 significantly enriched pathways from the MSigDB Hallmark (H) collection. The x-axis represents the normalized enrichment score (NES). Positive NES values indicate pathways enriched in the [knockdown group], while negative NES values indicate pathways enriched in the [control group]. The bar colors are categorized based on the Benjamini-Hochberg adjusted p-value. Only pathways with FDR-adjusted p value (q value) < 0.05 are shown (up to 20 pathways).

Article Snippet: 8 h: Total RNA from knockdown (KD) and negative control (NC) cells was submitted to Plasmidsaurus for 3’-RNA-seq.

Techniques: Gene Expression, Knockdown, Transformation Assay, Labeling, Comparison, Control