squamous cell carcinomas  (ATCC)

Journal: Journal of Dental Sciences

Article Title: APOBEC3B/ASF1B–TGF-β signaling axis promotes epithelial–mesenchymal transition in HPV-positive oropharyngeal cancer

doi: 10.1016/j.jds.2025.10.039

Figure Lengend Snippet: Functional characterization of APOBEC3B silencing and its downstream effects in vitro. (A) DepMap analysis identifying HPV-negative (SCC-4) and HPV-positive (SCC-154) oral squamous cell carcinoma lines with differential APOBEC3B expression suitable for functional validation. (B) Quantitative PCR analysis showing that APOBEC3B silencing significantly downregulates its downstream targets ASF1B and RPA2, indicating disruption of the APOBEC3B/ASF1B regulatory axis. (C–D) Cisplatin sensitivity, migration, and invasion assays demonstrate that APOBEC3B knockdown reduces cell motility and enhances cisplatin-induced cytotoxicity, as assessed by CellTiter-Glo viability assay (Promega) and Matrigel-based invasion analysis.(E) Tumor spheroid formation assay showing that shAPOBEC3B markedly suppresses both the number and size of tumor spheres in HPV-positive SCC-154 cells, indicating impaired self-renewal capacity and partial re-sensitization of irradiation-resistant cells to treatment.Data represent mean ± SD from three independent experiments; statistical significance was determined by Student's t-test ( P < 0.05). Abbreviations: SD, standard deviation; EMT, epithelial–mesenchymal transition; HPV, human papillomavirus; shRNA, short hairpin RNA.

Article Snippet: Human oropharyngeal squamous cell carcinoma cell lines SCC-4 (HPV-negative) and SCC-154 (HPV-positive) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, In Vitro, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Disruption, Migration, Knockdown, Viability Assay, Tube Formation Assay, Irradiation, Standard Deviation, shRNA

Journal: bioRxiv

Article Title: NF-κB mediates transactivation of HNRNPD, resulting in PTEN destabilization and constitutive activation of the PI3K-AKT pathway in oral cancer cells

doi: 10.1101/2025.01.05.631360

Figure Lengend Snippet: (A) Total RNA isolated from SCC-4 cells and SCC-4 hnRNPD KO clones was reverse transcribed and subjected to real time PCR using specific PTEN primers. Simultaneously, the PCR was also performed using 18S ribosomal RNA specific primers and served as internal control for normalization of PTEN transcript. (B) Schematic representation depicting the positions of hnRNPD binding motif in the PTEN 3’UTR and primers used for RIP assay. (C) The SCC-4 control and SCC-4 hnRNPD KO1 cells were treated with 5μg/ml of actinomycin D for various time points to block mRNA synthesis. Total RNA was harvested followed by the qPCR as described in the text. Half-life was calculated using non-linear one phase exponential decay equation inGraphpad Prism 6.0. (D) HnRNPD bound mRNA was immunoprecipitated from SCC-4 control and SCC-4 hnRNPD KO cells with anti-hnRNPD antibody and subjected PCR after elution of bound mRNA from hnRNPD using the primers depicted in the figure 24C. Total cellular RNA isolated from SCC-4 cells was used as input. PCR with gene specific primers and RNA immunoprecipitated using normal rabbit IgG served as negative control (IgG lane). Similarly, PCR performed with gene specific primers without a template also served as control. A 50 bp DNA ladder was used to determine the size of the amplified PCR fragments on agarose gel. Representative agarose gel image of RT-PCR fragment using RIP assay for hnRNPD binding motif on PTEN mRNA. Values are mean ± SD from three independent experiments. Values significantly different from respective controls have been marked by stars (**P ≤ 0.01, ****P ≤ 0.0001). Expression of PTEN and hnRNPD in oral cancer. (E) Paraffin-embedded tissue sections of oral cancer were immunostained using anti-hnRNPD or anti-PTEN antibody. The expressions of both the proteins were scored independently by two pathologists blinded to the identity of sections and their score. The representative images of OSCC immunostained sections. Nuclear expression of hnRNPD (I-II) in OSCC, whereas mild cytoplasmic staining of PTEN (III-IV). (F) Correlation between hnRNPD and PTEN expression in OSCC tissue specimens was assessed by Spearmen’s correlation analysis. This analysis revealed a strong negative correlation between nuclear expression of hnRNPD and cytoplasmic expression of PTEN in OSCC. Values are mean ± SD from three independent experiments. Values significantly different from respective controls have been marked by stars (**P ≤ 0.01, ****P ≤ 0.0001).

Article Snippet: Oral squamous cell carcinoma (OSCC) cell line SCC-4 was obtained from American Type Culture Collection (ATCC, VA, USA) and characterized by STR profiling.

Techniques: Isolation, Clone Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Binding Assay, Blocking Assay, Immunoprecipitation, Negative Control, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining