Journal: bioRxiv

Article Title: Eukaryotic Initiation Factor 5B (eIF5B)-Driven Translational Control Impacts Oral Squamous Cell Carcinoma Pathophysiology

doi: 10.64898/2026.01.30.702967

Figure Lengend Snippet: BJ-5ta cells were transfected with either a non-specific control siRNA (siC) or an eIF5B-specific siRNA pool (si-eIF5B). (A) Following 24 h of transfection, cells were treated with either vehicle control or TRAIL (100 ng/mL). Cell viability was assessed after an additional 72 h using alamarBlue TM assays. (B) Annexin V and 7-AAD staining and flow cytometric analysis of transfected cells. The top two panels, siC and siC + TRAIL, the bottom two panels, si5B and si5B + TRAIL, were harvested at 96 h post-transfection and treated with Annexin V + 7AAD. Contour plots are divided into quadrants Live (lower left; cells staining negative for both 7-AAD and annexin V), Q1 (upper left; cells staining positive for 7-AAD but negative for annexin V), Early apoptosis (lower right; staining annexin V positive and 7-AAD negative), and Late apoptosis (upper right; staining positive for both 7-AAD and annexin V). (C) Representative immunoblots probing for eIF5B, XIAP, Bcl-xL, cIAP1, cFLIPs, DR 4, DR 5, and β-actin (internal control) are shown. ( D) Control and eIF5B-depleted BJ-5ta cells were treated with the 4EGI-1 inhibitor to inhibit cap-dependent translation. Levels of XIAP were monitored under control and the 4EGI-1 treatment conditions. (E) eIF5B-bound RNA immunoprecipitations (RIPs) followed by qRT-PCR measurements of input-normalized tRNAiMet levels from BJ-5ta vs UMSCC-29. Data are presented as mean ± SEM from three independent biological replicates, with statistical significance indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CAL-33 and BJ-5ta (immortalized fibroblast) cell lines were obtained from American Type Culture Collection (ATCC; Virginia, USA), and UMSCC-1 and UMSCC-29 were obtained from the University of Michigan (Michigan, USA).

Techniques: Transfection, Control, Staining, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Eukaryotic Initiation Factor 5B (eIF5B)-Driven Translational Control Impacts Oral Squamous Cell Carcinoma Pathophysiology

doi: 10.64898/2026.01.30.702967

Figure Lengend Snippet: BrdU incorporation assay was used to measure cell proliferation. Quantification shows the percentage of BrdU-labeled cells in control (siC) and si5B-treated groups. Data are presented as mean ± SEM; ***p < 0.001. (A) UMSCC-29 (B) BJ-5ta (C) Collagen-based invasion assay was used to evaluate cell invasiveness in control (siC) and si5B-treated groups. Cells were seeded onto collagen matrices and allowed to invade for a defined period (48-72 hours). The number of invading cells was quantified and expressed as the mean ± SEM. Microscopy images of invading cells (C; left panel) , quantification (C; right panel) . Phase contrast microscopy images showing wound area in siC/si5B-treated OSCC cells at the time of scratch introduction (T = 0 h) & at T = 30 h wound closure (c; top panel) . Quantification of the percentage of wound area covered after 30 hours, normalized to wound area at the time of scratch introduction (c; lower panel) . (E) Representative images of the endothelial HUVEC cells tube formation assay treated with conditioned media from siC- or si5B-transfected OSCC. ImageJ angiogenesis macro software quantified Junctions, segments, and nodes. OSCC cells were transfected with either control siRNA (siC) or eIF5B-specific siRNA (si5B) and incubated for 96 hours. (F) The conditioned media for the control and eIF5B-depleted OSCC cells were analyzed for angiogenic biomarkers (lower panel) using EVE Technologies. (G) Representative images of immunoblots quantified probing for eIF5B, p-EGFR, EGFR, p-ERK, ERK, p-NF-kB, NF-kB, HIF1α, VEGFA, and β-actin (internal control). Data are presented as mean ± SEM from three independent biological replicates, with statistical significance indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001, and ****, p< 0.0001.

Article Snippet: CAL-33 and BJ-5ta (immortalized fibroblast) cell lines were obtained from American Type Culture Collection (ATCC; Virginia, USA), and UMSCC-1 and UMSCC-29 were obtained from the University of Michigan (Michigan, USA).

Techniques: BrdU Incorporation Assay, Labeling, Control, Invasion Assay, Microscopy, Tube Formation Assay, Transfection, Software, Incubation, Western Blot

Journal: bioRxiv

Article Title: Eukaryotic Initiation Factor 5B (eIF5B)-Driven Translational Control Impacts Oral Squamous Cell Carcinoma Pathophysiology

doi: 10.64898/2026.01.30.702967

Figure Lengend Snippet: BJ-5ta cells were transfected with either control siRNA (siC) or eIF5B-specific siRNA (si5B) and incubated for 96 hours. TRAIL (100 ng/mL) treatment was performed for 4 hours, then the media was collected. The conditioned media for the control and eIF5B-depleted BT5Ta cells were analyzed for angiogenic biomarkers using EVE Technologies. The level of distinct angiogenic biomarkers was decreased in the spent media of eIF5B-depleted BJ-5ta cells. Data are expressed as mean ± SEM for three biological replicates. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: CAL-33 and BJ-5ta (immortalized fibroblast) cell lines were obtained from American Type Culture Collection (ATCC; Virginia, USA), and UMSCC-1 and UMSCC-29 were obtained from the University of Michigan (Michigan, USA).

Techniques: Transfection, Control, Incubation

Journal: bioRxiv

Article Title: Reversibility of Nuclear and 3D Genomic Changes in Non-Cancerous Fibroblasts After Constricted Migration

doi: 10.64898/2026.01.28.701973

Figure Lengend Snippet: A) Schematic of BJ-5ta Transwell migration assay. B) Quantification of migration rates (% of cells that migrated through pores) through 5 µm pores for BJ-5ta cells across 5 rounds of sequential migration. C) Confocal images (63x) of BJ-5ta nuclei stained with LaminA/C (green) and DAPI (blue) for both unmigrated conditions and sequential migration rounds (R1-R4). Scalebar = 10 µm. Aspect Ratio (D) , Solidity (E) and EFC Ratio (F) measurements for conditions shown in 1C . (****P<.0001, ***P<.001, **P<.01, *P<.05, one-way ANOVA. N = 60-250 nuclei per condition.) G) Cumulative distribution plot of LaminA/C standard deviation for conditions shown in 1C . N = 60-250 nuclei per condition. In all violin plots, thick dashed lines indicate median, and thin dashes indicate 25 th and 75 th percentiles, respectively.

Article Snippet: BJ-5ta Cells were purchased from ATCC (CRL-4001).

Techniques: Transwell Migration Assay, Migration, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Reversibility of Nuclear and 3D Genomic Changes in Non-Cancerous Fibroblasts After Constricted Migration

doi: 10.64898/2026.01.28.701973

Figure Lengend Snippet: A) Confocal images (63x) of BJ-5ta nuclei stained with Lamin A/C (green) and DAPI (blue) of S/S and R2 immediately after migration (no growth) and then followed through 4 days of proliferation. Scalebar = 10 µm. Aspect Ratio (B) , Solidity (C) and EFC Ratio (D) measurements for conditions shown in 2A . (****P<.0001, ***P<.001, **P<.01, *P<.05, one-way ANOVA. N = 68-204 nuclei per condition.) In all violin plots, thick dashed lines indicate median, and thin dashes indicate 25 th and 75 th percentiles, respectively.

Article Snippet: BJ-5ta Cells were purchased from ATCC (CRL-4001).

Techniques: Staining, Migration