Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: ALDH2 functions as a tumor suppressor in vitro. (A-D) Quantitative RT-PCR and immunoblot analyses were performed to evaluate ALDH2 mRNA and protein levels. Actin beta ( ACTB ) and tubulin alpha-1B chain (TUBA1B) served as internal controls for quantitative RT-PCR and immunoblotting, respectively. (A) Endogenous ALDH2 mRNA and (B) ALDH2 protein levels in four HNSC-derived cell lines. (C) Stable knockdown of ALDH2 in Detroit 562 and SCC-25 cells using replication-incompetent lentiviruses carrying two distinct shRNA clones targeting ALDH2 . (D) Stable overexpression of ALDH2 in 2A3 and FaDu cells using lentiviruses packaged from pLenti-ALDH2-Myc-DDK-P2A-Puro plasmid. (E-P) Functional assays, including soft agar colony formation and MTT viability assays, immunoblotting, transwell migration and invasion, ELISA, and the tube formation assay using HUVECs, were conducted following ALDH2 knockdown or overexpression in the four cell lines. (E) Anchorage-independent cell growth. (F) Expression levels of cell cycle-related proteins. (G) Cell migration and invasion. (H) Expression levels of epithelial-mesenchymal transition (EMT) markers. (I) VEGFA levels in conditioned media. (J) In vitro angiogenesis in HUVECs treated with conditioned media from ALDH2 -knockdown Detroit 562 and SCC-25 cells. (K-P) Opposing phenotypes observed in ALDH2 -overexpressing 2A3 and FaDu cells, confirmed by functional assays, immunoblotting, and ELISA. Data are presented as mean ± SD. Statistical significance: * p < 0.05, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Derivative Assay, Knockdown, shRNA, Clone Assay, Over Expression, Plasmid Preparation, Functional Assay, Migration, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: ALDH2 suppresses tumorigenesis in vitro by inhibiting the RAS-AKT signaling. (A , B) Immunoblot analysis of RAS-AKT pathway components, including NR4A1, in ALDH2 -knockdown Detroit 562 and ALDH2 -overexpressing FaDu cells. (C) RAS pull-down activation assay using Raf-RBD glutathione beads, selectively captured GTP-bound RAS (active form), followed by immunoblotting with pan-RAS antibody to assess RAS activation status in ALDH2 -knockdown Detroit 562 and ALDH2-overexpressing FaDu cells. (D) Schematic of the experimental design of co-overexpression of ALDH2 and HRAS(G12D) in 2A3 and FaDu cells. (E) Transient transfection of pHRAS(G12D)-HaloTag plasmid induced expression of the HRAS(G12D)-HaloTag fusion protein (~ 54 kDa) in 2A3 and FaDu cells. (F-H) Functional assays, including soft agar colony formation, MTT viability, transwell migration and invasion, and HUVEC tube formation (calcein AM staining, quantified via ImageJ) were performed using conditioned media from four groups: Lenti/pHaloTag (control), ALDH2-/pHaloTag-transfected, Lenti/pHRAS(G12D)-transfected, and ALDH2-/pHRAS(G12D)-transfected cells. (I) Immunoblot analysis of RAS-AKT pathway proteins, including NR4A1, following ALDH2 and/or HRAS(G12D) overexpression in 2A3 and FaDu cells. (J) HRAS protein and HRAS mRNA levels were assessed by immunoblot and quantitative RT-PCR after HRAS knockdown using three distinct shRNA clones in Detroit 562 and ALDH2 -knockdown Detroit 562 cells. (K) Schematic of a dual-knockdown strategy targeting ALDH2 and HRAS in Detroit 562 and SCC-25 cells. (L-N) Functional assays, including soft agar/MTT, transwell migration and invasion, and HUVEC tube formation by treatments with conditioned media from HRAS -knockdown and ALDH2/HRAS dual knockdown Detroit 562 and SCC-25 cells. (O) Immunoblot analysis of RAS-AKT pathway components, including NR4A1, in Detroit 562 and SCC-25 cells following HRAS -knockdown and ALDH2/HRAS dual knockdown. Data are presented as mean ± SD. Statistical significance: ** p < 0.01, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: In Vitro, Western Blot, Knockdown, Activation Assay, Over Expression, Transfection, Plasmid Preparation, Expressing, Functional Assay, Migration, Staining, Control, Quantitative RT-PCR, shRNA, Clone Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: ALDH2 transactivates TGM2 to induce apoptosis in HNSC-derived cells. (A) Immunoblotting and CASP3/7 activity assay (colorimetric) assessed apoptosis-related protein levels and caspase activity in ALDH2 -knockdown Detroit 562 and ALDH2-overexpressing FaDu cells. (B , C) Quantitative RT-PCR and immunoblot analysis measured TGM2 mRNA and protein levels in ALDH2-overexpressing 2A3 and FaDu cells, and ALDH2 -knockdown Detroit and SCC-25 cells. (D) Apoptotic cells were quantified via flow cytometry using annexin V-FITC/PI staining after 72 h of transfection with pTGM2-HaloTag plasmids. (E) TGM2 promoter activity was assessed using the Dual-Luciferase ® Reporter Assay with pGL4.17 (control), pGL4.17-A TGM2 (−900 to +100 ), and pGL4.17-B TGM2 (−600 to +100) constructs cotransfected with pRL Renilla vector (14:1) into Detroit 562, ALDH2-overexpressing FaDu, and ALDH2 -knockdown Detroit cells. (F , G) Immunoblot analysis was performed to evaluate TGM2 protein levels in 2A3 and FaDu cell lysates from Fig. D, encompassing Lenti/pHaloTag, HaloTag/ALDH2 overexpression, Lenti/HRAS(G12D)-HaloTag overexpression, and dual overexpression of ALDH2/HRAS(G12D)-HaloTag, and from Fig. k, containing shLuc, shALDH2#2, shHRAS#3, and double knockdown shALDH2#2/shHRAS#3. (H , I) Stable overexpression of ALDH2(WT), ALDH2(E504K), ALDH2(T261A), ALDH2(S488A), and ALDH2(T261A/S488A) was achieved via transduction of replication-incompetent lentiviral vectors carrying the specific target gene into FaDu and ALDH2 -knockdown Detroit 562 cells. TGM2 mRNA and protein levels were measured using quantitative RT-PCR and immunoblot analysis, respectively. (J-O) Functional assays following TGM2 overexpression in ALDH2 -knockdown Detroit 562 cells. (J) Experimental design. (K) Immunoblotting confirmed pTGM2-HaloTag transfection and expression of ~ 110 kDa fusion protein. (L-N) Soft agar/anchorage-independent cell growth/MTT, transwell migration and invasion, and HUVEC tube formation (calcein AM staining/ImageJ) using conditioned media were assessed from control (shLuc/pHaloTag), shLuc/pTGM2-HaloTag, shALDH2#2/pHaloTag, and shALDH2#2/pTGM2-HaloTag groups. (O) Flow cytometry with annexin V-FITC/PI staining evaluated apoptosis across groups. (P) Immunblotting and CASP3/7 assay quantified apoptosis-related proteins and caspase activity. (Q) TGM2 knockdown in SCC-25 cells using three shRNA clones was validated by quantitative RT-PCR and immunoblotting. (R-W) Functional and apoptosis marker analyses were performed in shLuc/Lenti (control), shLuc/ALDH2-overexpressing, shTGM2#2/Lenti, and shTGM2#2/ALDH2-overexpressing SCC-25 cells. Data are presented as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: Derivative Assay, Western Blot, Activity Assay, Knockdown, Quantitative RT-PCR, Flow Cytometry, Staining, Transfection, Luciferase, Reporter Assay, Control, Construct, Plasmid Preparation, Over Expression, Transduction, Functional Assay, Expressing, Migration, shRNA, Clone Assay, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: Cooperative interaction between ALDH2 and ALDH6A1 enhances suppression of tumorigenetic traits in vitro. (A) Immunoblotting revealed ALDH6A1 protein expression across various HNSC-derived cell lines. Notably, transfection with the pALDH6A1 plasmid for 48 h led to a marked increase in ALDH6A1-HaloTag fusion protein (~ 91 kDa) in ALDH2-overexpressing 2A3 and FaDu cells. (B) ALDH6A1-HaloTag fusion protein levels were notably elevated following pALDH6A1-HaloTag transfection in ALDH2-overexpressing 2A3 and FaDu cells. (C-F) 2A3 and FaDu cells were subjected to the following transfection conditions: control (Lenti/pHaloTag), ALDH2 overexpression (ALDH2/pHaloTag), ALDH6A1 overexpression (Lenti/pALDH6A1-HaloTag), and co-overexpression of ALDH2 and ALDH6A1 (ALDH2/pALDH6A1-HaloTag). A series of functional assays was used to analyze the phenotypic alterations, including anchorage-independent cell growth (soft agar colony formation/MTT), cell motility and invasiveness (transwell migration and invasion), and angiogenic potential (HUVEC tube formation with calcein AM staining following treatments with conditioned media collected from different groups). (G-J) Detroit 562 and SCC-25 cells were transfected with pALDH6A1-HaloTag plasmids under ALDH2 -knockdown conditions to assess the phenotypic impact of ALDH6A1 overexpression. Four experimental groups were established: control (shLuc/pHaloTag), ALDH6A1 overexpression (shLuc/pALDH6A1-HaloTag), ALDH2 knockdown (shALDH2#2/pHaloTag), and ALDH6A1 overexpression in ALDH2 -knockdown background (shALDH2#2/pALDH6A1-HaloTag). Functional assays were performed to evaluate anchorage-independent cell growth via soft agar colony formation/MTT, cell motility, and invasiveness using transwell migration and invasion assays, followed by calcein AM staining after treatment with conditioned media from each group. (K) Protein-protein docking using ClusPro (Piper algorithm) predicted interaction interfaces between ALDH2 and ALDH6A1. PyMOL visualization identified contact residues: ALDH2 (green) and ALDH6A1 (cyan), with atoms color-coded (red: oxygen, blue: nitrogen, yellow: sulfur, white: hydrogen). (L) PyMOL further highlighted polar interactions using yellow dashed lines. (M) Pull-down assays employing anti-ALDH2 antibodies, followed by immunoblotting with anti-HaloTag, confirmed physical interaction between ALDH2 and ALDH6A1 in ALDH2-overexpressing FaDu cells transfected with pALDH6A1-HaloTag. Mutually, pull-down using anti-HaloTag antibodies, followed by immunoblotting with anti-ALDH2, further validated this interaction. (N) Reciprocal co-immunoprecipitation (Co-IP) assays using anti-ALDH2 and anti-ALDH6 antibodies validated endogenous interaction in Detroit 562 and SCC-25. Anti-GFP probing served as a negative control. (O , P) Immunoblot analysis confirmed expression of the ALDH6-HaloTag fusion protein (~ 91 kDa) following transient transfection of pALDH6A1-HaloTag plasmids into FaDu and shALDH2#2 Detroit 562 cells overexpressing ALDH2(WT) or one of five ALDH2 mutants. (Q) Reciprocal pull-down assays using anti-HaloTag and anti-ALDH2 antibodies confirmed that ALDH6A1 interacts with the E504K and phosphorylation-deficiency mutants, similar to its interaction with ALDH2(WT). Data are presented as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: In Vitro, Western Blot, Expressing, Derivative Assay, Transfection, Plasmid Preparation, Control, Over Expression, Functional Assay, Migration, Staining, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Phospho-proteomics

Journal: Radiology and Oncology

Article Title: Human papillomavirus-related oropharyngeal squamous cell carcinoma exhibits enhanced radiosensitivity despite limited activation of cytosolic DNA sensing pathways and innate immune responses

doi: 10.2478/raon-2025-0057

Figure Lengend Snippet: Baseline expression of cytosolic DNA sensors and cytokines in tumor cells and tumor microenvironment (TME) of pharyngeal squamous cell carcinomas (PSCCs). (A) Relative gene expression of cytosolic DNA sensors in cells in vitro , normalized to housekeeping genes (GUSB and B2M) (n = 3). (B) Relative gene expression of cytosolic DNA sensors in tumor cells in vivo , normalized to housekeeping genes (GUSB and B2M) (n = 5). (C) Relative gene expression of cytosolic DNA sensors in TME, normalized to housekeeping genes (BA and GADP) (n = 5). (D) Relative gene expression of cytokines in cells in vitro , normalized to housekeeping genes (GUSB and B2M) (n = 3). (E) Relative gene expression of cytokines in tumor cells in vivo , normalized to housekeeping genes (GUSB and B2M) (n = 5). (F) Relative gene expression of cytokines in TME, normalized to housekeeping genes (BA and GADP) (n = 5). Data is represented as mean ± standard error of the mean (SEM). # = indicates p < 0.05 for comparisons between UPCI:SCC090 and other cell lines or tumor models; * = indicates p < 0.05 for comparisons between irradiation (IR) doses within the same cell line or tumor model; o = indicates p < 0.05 for comparisons between FaDu and 2A3 models; + = indicates p < 0.05 for comparisons between UM-SCC-6 and FaDu

Article Snippet: The in vitro experiments were performed with four human PSCC cell lines: HPV16-related OPSCC UPCI:SCC090 (RRID:CVCL_1899; ATCC ® CRL-3239TM, Manassas, VA, USA), HPV16-unrelated OPSCC UM-SCC-6 (RRID:CVCL_7773; Merck-Millipore, Burlington, MA, USA), HPV16-related hypopharyngeal squamous cell carcinoma (HPSCC) 2A3 (RRID:CVCL_0D71; ATCC ® CRL-3212TM, ATCC), and HPV16-unrelated HPSCC FaDu (RRID:CVCL_1218; ATCC ® HTB-43TM, ATCC).

Techniques: Expressing, Gene Expression, In Vitro, In Vivo, Irradiation

Journal: Radiology and Oncology

Article Title: Human papillomavirus-related oropharyngeal squamous cell carcinoma exhibits enhanced radiosensitivity despite limited activation of cytosolic DNA sensing pathways and innate immune responses

doi: 10.2478/raon-2025-0057

Figure Lengend Snippet: Irradiation (IR)-induced accumulation of dsDNA in the cytosol of pharyngeal squamous cell carcinoma (PSCC) cells. (A) Accumulation of dsDNA in the cytosol of cells 72 hours after IR. Green: dsDNA (anti-dsDNA), red: plasma membrane (WGA), blue: nucleus (Hoechst 33342), Scale bar = 10 μm. (B) Number of dsDNA spots per cell in cytosol 72 hours after IR with 4, 8, or 3x8 Gy (n = 8). Data is represented as mean ± standard error of the mean (SEM). # = indicates p < 0.05 for comparisons between UPCI:SCC090 and other cell lines or tumor models; * = indicates p < 0.05 for comparisons between IR doses within the same cell line or tumor model; o = indicates p < 0.05 for comparisons between FaDu and 2A3 models; + = indicates p < 0.05 for comparisons between UM-SCC-6 and FaDu

Article Snippet: The in vitro experiments were performed with four human PSCC cell lines: HPV16-related OPSCC UPCI:SCC090 (RRID:CVCL_1899; ATCC ® CRL-3239TM, Manassas, VA, USA), HPV16-unrelated OPSCC UM-SCC-6 (RRID:CVCL_7773; Merck-Millipore, Burlington, MA, USA), HPV16-related hypopharyngeal squamous cell carcinoma (HPSCC) 2A3 (RRID:CVCL_0D71; ATCC ® CRL-3212TM, ATCC), and HPV16-unrelated HPSCC FaDu (RRID:CVCL_1218; ATCC ® HTB-43TM, ATCC).

Techniques: Irradiation, Clinical Proteomics, Membrane

Journal: Radiology and Oncology

Article Title: Human papillomavirus-related oropharyngeal squamous cell carcinoma exhibits enhanced radiosensitivity despite limited activation of cytosolic DNA sensing pathways and innate immune responses

doi: 10.2478/raon-2025-0057

Figure Lengend Snippet: Effect of irradiation (IR) on cytokine gene expression in tumor cells and tumor microenvironment (TME) of pharyngeal squamous cell carcinoma (PSCC). (A–C) Fold change in expression of IFNβ (A) , tumor necrosis factor (TNF)α (B) , and IL1β (C) in vitro 72 hours after IR with 4, 8, or 3x8 Gy, normalized to housekeeping genes (GUSB and B2M) and respective controls (n = 3). (D–F) Fold change in expression of IFNβ (D) , TNFa (E) , and IL1 β (F) in tumor cells in vivo 72 hours after IR with 8 or 3×8 Gy, normalized to housekeeping genes (GUSB and B2M) and control (n = 5). (G–I) Fold change in expression of Ifnβ (G) , TNFα (H) , and Il1β (I) in the TME in vivo 72 hours after IR with 8 or 3–8 Gy, normalized to housekeeping genes (BA and GADPH) and control (n = 5). Data is represented as mean ± standard error of the mean (SEM). # = indicates p < 0.05 for comparisons between UPCI:SCC090 and other cell lines or tumor models; * = indicates p < 0.05 for comparisons between IR doses within the same cell line or tumor model; o = indicates p < 0.05 for comparisons between FaDu and 2A3 models; + = indicates p < 0.05 for comparisons between UM-SCC-6 and FaDu

Article Snippet: The in vitro experiments were performed with four human PSCC cell lines: HPV16-related OPSCC UPCI:SCC090 (RRID:CVCL_1899; ATCC ® CRL-3239TM, Manassas, VA, USA), HPV16-unrelated OPSCC UM-SCC-6 (RRID:CVCL_7773; Merck-Millipore, Burlington, MA, USA), HPV16-related hypopharyngeal squamous cell carcinoma (HPSCC) 2A3 (RRID:CVCL_0D71; ATCC ® CRL-3212TM, ATCC), and HPV16-unrelated HPSCC FaDu (RRID:CVCL_1218; ATCC ® HTB-43TM, ATCC).

Techniques: Irradiation, Gene Expression, Expressing, In Vitro, In Vivo, Control

Journal: Radiology and Oncology

Article Title: Human papillomavirus-related oropharyngeal squamous cell carcinoma exhibits enhanced radiosensitivity despite limited activation of cytosolic DNA sensing pathways and innate immune responses

doi: 10.2478/raon-2025-0057

Figure Lengend Snippet: Response of the cellular innate immune system to irradiation (IR). (A) Frozen tumor sections were stained with anti-CD31 (red, Alexa 647), anti-F4/80 (green, Alexa 488), anti-natural killer cells (NK)p46 (orange, Cyanine 3), and Hoechst 33342 (blue). Scale bar: 100 μm. (B) Percentage of macrophages in tumor models before and after IR was determined by anti-F4/80 (calculated as the number of macrophages divided by the number of tumor cells). (C) Percentage NK cells in tumor models before and after IR was determined by anti-NKp46 (calculated as the number of NK divided by the number of tumor cells). Data are presented as mean ± standard error of the mean (SEM). # = indicates p < 0.05 for comparisons between UPCI:SCC090 and other cell lines or tumor models; * = indicates p < 0.05 for comparisons between IR doses within the same cell line or tumor model; o = indicates p < 0.05 for comparisons between FaDu and 2A3 models

Article Snippet: The in vitro experiments were performed with four human PSCC cell lines: HPV16-related OPSCC UPCI:SCC090 (RRID:CVCL_1899; ATCC ® CRL-3239TM, Manassas, VA, USA), HPV16-unrelated OPSCC UM-SCC-6 (RRID:CVCL_7773; Merck-Millipore, Burlington, MA, USA), HPV16-related hypopharyngeal squamous cell carcinoma (HPSCC) 2A3 (RRID:CVCL_0D71; ATCC ® CRL-3212TM, ATCC), and HPV16-unrelated HPSCC FaDu (RRID:CVCL_1218; ATCC ® HTB-43TM, ATCC).

Techniques: Irradiation, Staining

Journal: Bio-protocol

Article Title: Dual Phospho-CyTOF Workflows for Comparative JAK/STAT Signaling Analysis in Human Cryopreserved PBMCs and Whole Blood

doi: 10.21769/BioProtoc.5512

Figure Lengend Snippet: The sequential gating strategy was used to identify immune cell populations from healthy human samples. Healthy donor samples, either ( A ) cryopreserved peripheral blood mononuclear cells (cPBMCs) or ( B ) fresh whole blood (WB), were stimulated for 15 min, fixed, palladium-barcoded, and stained with a comprehensive panel of 19 (for cPBMCs) or 20 (for whole blood) surface markers. Within the single-cell gate, non-granulocytes (leukocytes) were defined as CD66b-CD45 + , while granulocytes were confirmed solely in whole blood as CD66b + CD45-. Lymphocytes were resolved using CD3 and CD56 [T cells (CD3 + CD56-)], NKT cells (CD3 + CD56 + ), and NK cells (CD3-CD56 + ). B cells were identified as CD19 + CD20 +/- within the CD3-CD56- gate, further confirmed by negative expression of CD123 and CD11c. Both T and B cells were further subset based on the expression of CD4, CD8, CD45RA, CD27, and CD25 or IgD and CD27, respectively. Non-T, non-B, and non-NK cells (CD45 + CD3-CD19-CD56-CD20-) were separated based on their expression of CD11c and HLA-DR. Total DCs were defined as CD11c + HLA-DR + and can be further defined through the expression of CD123 (plasmacytoid DCs). Monocytes were resolved within this non-lymphocyte gate based on their expression of CD14 and CD16.

Article Snippet: 149 Sm , CD25 (IL-2R) , Standard BioTools , 3149010B , 2A3 , 43.75.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A-D) Venn diagrams of differentially expressed genes in FaDu (A-B) and 2A3 (C-D) cell lines. Venn diagrams illustrate the number of significantly regulated genes under three conditions: hypoxia (green), irradiation (yellow), and the combination of hypoxia and irradiation (blue). Numbers in the overlapping areas indicate genes commonly regulated under the corresponding conditions. Genes were considered differentially expressed if they had an adjusted p-value (padj) <0.1. (E) Biological processes differentially regulated in FaDu and 2A3 HNSCC cell lines under hypoxia and combined hypoxia with gamma irradiation. The figure illustrates biological processes significantly upregulated (red) or downregulated (green) in FaDu and 2A3 cells in response to hypoxia alone or in combination with gamma radiation. The analysis was performed using the WebGestalt 2024 platform and included only genes that were consistently detected across all three independent biological replicates and met the inclusion criteria of adjusted p < 0.1 and |log 2 FC| ≥ 2.

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Irradiation

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A) FaDu, 2A3, and Detroit-562 cell lines were cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Cell numbers were determined 48 and 72 hours after plating, and doubling times were calculated as described in Methods. Data represent the mean ± SD from four independent experiments (n = 4). Statistical analysis was performed using an unpaired t-test (**p < 0.01). (B) Heatmap of the top 20 DEGs. Averaged data from three independent experiments are shown. The color bar is showing the values of z-score for each gene after library size normalization via DESeq2 software. Cell cycle distribution was analyzed 24 hours after 6 Gy gamma-irradiation in (C) FaDu, (D) 2A3, and (E) Detroit-562 cells cultured under normoxic or hypoxic (1% O 2 ) conditions. Results are presented as the percentage of cells in G0/1, S, or G2/M phases. Data represent the mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Cell Culture, Software, Irradiation

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A) Cleaved caspase-3 levels in FaDu, 2A3, and Detroit-562 cells cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, assessed 48 hours after 6 Gy gamma-irradiation. Results are shown as fold change relative to non-irradiated normoxic controls. Data represent mean ± SD from three independent experiments (n = 3). Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001. (B) Heatmap of the top 20 DEGs. Averaged data from three independent experiments are shown. The color bar is showing the values of z-score for each gene after library size normalization via DESeq2 software. (C) Representative western blots of N-cadherin, E-cadherin, and vimentin in FaDu and 2A3 cells. (D) Quantification of protein levels expressed as mean optical density (O.D.) ± SD, normalized to β-actin, in FaDu and 2A3 cells under normoxic or hypoxic conditions. (E–G) Effects of hypoxia and irradiation on N-cadherin, E-cadherin, and vimentin levels in FaDu and 2A3 cells under normoxic and hypoxic conditions. Data represent mean ± SD from three independent experiments (n = 3). Statistical analysis: unpaired t-test (*p < 0.05).

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Cell Culture, Irradiation, Software, Western Blot

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A) Heatmap of the top 20 DEGs. Averaged data from three independent experiments are shown. The color bar is showing the values of z-score for each gene after library size normalization via DESeq2 software. (B) Cytokine/chemokine production in response to hypoxia, gamma-irradiation, and their combination. Conditioned media from FaDu, 2A3, and Detroit-562 cells were collected 48 hours after gamma-irradiation under normoxic or hypoxic conditions. Media from three independent experiments per cell line were pooled for analysis. Results are presented as z-scores of log 10 -transformed normalized data. n.d. = not detected. (C) Cell numbers of FaDu, 2A3, and Detroit-562 cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, assessed 48 hours after 6 Gy gamma-irradiation. Data represent mean ± SD from four independent experiments (n = 4). Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001. Levels of IL-8 (D), MIF (E), and Serpin E1 (F) in supernatants from FaDu, 2A3, and Detroit-562 cells cultured under normoxic or hypoxic conditions, assessed 48 hours after gamma-irradiation. Concentrations were normalized to cell numbers per condition. Data represent mean ± SD from three to four independent experiments (n = 3–4). Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Software, Irradiation, Transformation Assay, Cell Culture