5737s  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Oncogenic and immunomodulatory functions of SUV420H1 in HPV-negative head and neck squamous cell carcinoma

doi: 10.1101/2025.10.20.683419

Figure Lengend Snippet: (A) Relative SUV420H1 mRNA expression levels in the immortalized squamous epithelial cell line BEAS-2B and 5 HPV-negative HNSCC cell lines (PE/CA-PJ15, FaDu, HN-SCC-151, HN13, HN-6). Data are presented as mean ± SEM from n = 3 biological replicates. Ordinary one-way ANOVA with multiple comparisons; * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001. (B) CCK8 assays in 5 HPV-negative HNSCC cell lines treated with negative control (siNC) or three SUV420H1 -targeting siRNAs (si1, si2, si3) for 10-12 days. Data are presented as mean ± SEM from n=5 biological replicates. Student t-test; * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001. (C) Colony forming assays (CFAs) in 5 HPV-negative HNSCC cell lines treated with negative control (siNC) or three SUV420H1 -targeting siRNAs (si1, si2, si3) for 10 days. (D) Top: Western blotting for H4K20me3 levels in HN-SCC-151, HN13 and HN-6 cells treated with control DMSO or incremental concentrations of the SUV420H1 inhibitor A-196 for 6 days. Nuclear extracts were obtained and 5ug were loaded. H3 was used as a loading control. Bottom: Densitometry for H4K20me3 bands, normalized by H3 band intensity. Data are shown as mean ± SEM from n=2 independent biological replicates. (E) CCK8 assays in 5 HPV-negative HNSCC cell lines treated with control DMSO or A-196 at 0.5uM, 1uM and 2.5uM for 12 days. Data are presented as mean ± SEM from n=5 biological replicates. Student t-test; * p<0.05, ** p<0.01, *** p<0.005. (F) Colony forming assays (CFAs) in 5 HPV-negative HNSCC cell lines treated with control DMSO or A-196 at 0.5uM, 1uM and 2.5uM for 12 days. (G) Top: Bar graphs of quantified results of CCK8 assay after 10 days of treatment of 5 HPV-negative HNSCC cell lines with SUV420H1 -targeting siRNAs (left) or with A-196 (right). Data are presented as mean ± SEM from n=5 biological replicates. Bottom: Bar graphs of quantified results of CFAs after 10 days of treatment of 5 HPV-negative HNSCC cell lines with SUV420H1 -targeting siRNAs (left) or with A-196 (right). Data are presented as mean ± SEM from n=3 biological replicates. Student t-test; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (H) Top: Western blotting for H4K20me3, H4K20me2 and H4K20me1 in wild-type HN-6 parental cells and SUV420H1 -mutant cell lines Mut-1 (H273K) and Mut-2 (C275A). 5ug of nuclear extracts were loaded. H3 was used as a loading control. Two biological replicates are presented. Bottom: Densitometry for H4K20me3 bands, normalized by H3 band intensity. Data are shown as mean ± SEM from n=2 biological replicates. (I) CCK8 assay with parental wild-type HN-6 cells and SUV420H1 -mutant cell lines Mut-1 and Mut-2. Data are shown as mean ± SEM from n=5 biological replicates. (J) Top: CFAs of wild-type HN-6 parental cells and SUV420H1 -mutant cell lines Mut-1 and Mut-2. Bottom: Bar graphs of quantified results of CFAs. Data are presented as mean ± SEM from n=5 biological replicates.

Article Snippet: Then, 0.5ug of antibody targeting H4K20me3 (CST, 5737S) was added to each designated experimental tube.

Techniques: Expressing, Negative Control, Western Blot, Control, CCK-8 Assay, Mutagenesis

Journal: bioRxiv

Article Title: Oncogenic and immunomodulatory functions of SUV420H1 in HPV-negative head and neck squamous cell carcinoma

doi: 10.1101/2025.10.20.683419

Figure Lengend Snippet: (A) GSEA analysis of significantly enriched Hallmark pathways in HN-SCC-151 cells treated with si SUV420H1 (#1)/siNC (left) or A-196/DMSO (right) for 6 days. (B) Genomic coordinate heatmap of decreased/lost intragenic H4K20me3 peaks (n=3,322) in HN-SCC-151 cells treated with A-196 vs DMSO for 6 days. FDR<0.1, log2FC>0.38. Concordant RNA-seq heatmap of 2,017 corresponding genes with decreased/lost intragenic H4K20me3 peaks and available mRNA expression data; FDR<0.1. (C) Venn diagram of overlapping EMT and immune-response (IFNA/G-, inflammatory-response) Hallmark pathway genes and the 2,017 genes with decreased/lost H4K20me3 peaks and detectable mRNA expression (A-196 CUT&RUN and RNA-seq datasets). (D) Volcano plots of H4K20me3 peaks overlapping with EMT (127 peaks) or IFNA/G-(100 peaks) and inflammatory-response genes (122 peaks) (promoter and gene body regions only). FDR<0.1, LFC>0.38. (E) RNA-seq heatmaps of EMT, IFNA/G-response and inflammatory-response genes with decreased/lost H4K20me3 peaks and differential mRNA expression changes (FDR<0.1). (F) Top 10 putative EMT and immune-response gene targets with the “deepest” mRNA expression changes regulated by SUV420H1-mediated H4K20me3. (G) UCSC tracks for H4K20me3 obtained with CUT&RUN for H4K20me3 in HN-SCC-151 cells treated with DMSO or A-196 for 6 days. Representative EMT and immune-response genes with decreased/lost H4K20me3 after treatment with A-196 are shown.

Article Snippet: Then, 0.5ug of antibody targeting H4K20me3 (CST, 5737S) was added to each designated experimental tube.

Techniques: RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Oncogenic and immunomodulatory functions of SUV420H1 in HPV-negative head and neck squamous cell carcinoma

doi: 10.1101/2025.10.20.683419

Figure Lengend Snippet: (A) Suv420h1 knockout (KO) MOC1 cell lines generated with CRISPR. Top: Colony formation assays with control (NT) and two Suv420h1 KO MOC1 cell lines (KO-1, KO-2). The graph represents the average number of colonies per condition. Data are shown as mean ± SEM from n=3 independent biological replicates. Bottom: Western blotting for H4k20me3 in control and Suv420h1 KO MOC1 cell lines. Data are shown as mean ± SEM from n=2 biological replicates. (B) C57BL/6 mice were implanted with flank NT or Suv420h1 KO-1 or KO-2 MOC1 tumors and tumor volumes were measured over 28 days. Top graph: Average tumor volume (cm3). Data are shown as mean ± SEM. Two-way ANOVA test; ** p<0.01, *** p<0.001. Bottom graph: hairline tumor growth curves. (C) Multicolor flow cytometry analysis of wild-type and Suv420h1 KO MOC1 tumors. Data are represented as mean ± SEM. Unpaired t-test between NT and KO-1 or NT and KO-2; * p<0.05, ** p<0.01, *** p<0.001. (D) Average tumor volume (cm3) measured over 24 days in C57BL/6 mice bearing flank tumors from four experimental groups: control (NT), NT treated with anti-PD-1 antibody (NT+anti-PD-1), Suv420h1 KO-1, and Suv420h1 KO-1 treated with anti-PD-1 (KO-1+anti-PD-1). Two-way ANOVA test; p=0.05, **** p<0.0001. (E) Overall survival curves of four mouse groups: NT (n=7), NT+anti-PD-1 (n=8), Suv420h1 KO-1 (n=7), and Suv420h1 KO-1+anti-PD-1 (n=9). Survival was monitored for up to 80 days post-tumor implantation. Log-rank test; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Then, 0.5ug of antibody targeting H4K20me3 (CST, 5737S) was added to each designated experimental tube.

Techniques: Knock-Out, Generated, CRISPR, Control, Western Blot, Flow Cytometry, Tumor Implantation