Journal: Nanomaterials (Basel, Switzerland)

Article Title: In Vitro Analysis of Superparamagnetic Iron Oxide Nanoparticles Coated with APTES as Possible Radiosensitizers for HNSCC Cells.

doi: 10.3390/nano13020330

Figure Lengend Snippet: Figure 3. Intake of the SPIONs by the tumor cells. (a) Representative images of Cal33 cells, an HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC) cell line, with different concentrations of nanoparticles (0, 5, and 20 µg Fe/mL) over time (1, 24, 48, and 72 h). For the full course of the interaction and depiction of 50 µg Fe/mL, see the video in Supplementary Materials. (b) Immunofluorescence images of a cell from an HPV- HNSCC cell line RPMI 2650. Top to bottom: merge with DAPI (blue), α-Tubulin (green), nanoparticles (red); greyscale images of DAPI, α-Tubulin with orange line as estimated outline of the cell, nanoparticles captured with transmitted light microscope with red arrow on nanoparticles on cell margin, and white arrow on nanoparticles inside cell. (c) Three-dimensional immunofluorescence imaging of an RPMI 2650 cell with FITC-linked SPIONs (green), α-Tubulin (red), and DAPI (blue). (d) Orthogonal imaging of an RPMI 2650 cell with FITC-linked SPIONs (green), α-Tubulin (red), and DAPI (blue).

Article Snippet: Doubling times of the (c) RPMI 2650 and (f) Cal33 cells at doses from 0 to 8 Gy. (g) Exempl ry growth cu ves of cell line (BEAS-2B) with and without 20 μg Fe/mL SPIONs and at 0 and 2 Gy over 120 h. (h) Doubling times of the cell lines BEAS-2B, CLS 354, Det 562, HSC-4, UD-SCC-2, and UM-SCC-47, each with and without SPIONs at doses of 0 and 2 Gy. (i) Significances between the different groups of (h); error bars indicate the standard deviation.

Techniques: Light Microscopy, Imaging

Journal: Nanomaterials (Basel, Switzerland)

Article Title: In Vitro Analysis of Superparamagnetic Iron Oxide Nanoparticles Coated with APTES as Possible Radiosensitizers for HNSCC Cells.

doi: 10.3390/nano13020330

Figure Lengend Snippet: Figure 5. Detection of cytotoxic effects of SPION-APTES via colony formation assays. (a) Representa- tive images of stained BEAS-2B (healthy bronchial epithelium cell line) colonies in Petri dishes during colony formation assay; top: control, bottom: 2 Gy of ionizing radiation and 20 µg Fe/mL of SPIONs. Logarithmic plots of survival fraction of (b) RPMI 2650 and (c) Cal33 cells; control group without SPIONs (green) and treated group with 20 µg Fe/mL SPIONs (black); ionizing radiation doses from 0 to 8 Gy; * describes significance with p = 0.05 between control group and nanoparticle group at 8 Gy, determined by Mann–Whitney U-test. Logarithmic plots of surviving fraction at 0 and 2 Gy without SPIONs (green) and with 20 µg Fe/mL of SPIONs (black); dashed lines represent nanoparticle groups normalized to 1; cell lines (d) BEAS-2B; (e) Detroit 562 (Det 562); (f) HSC-4; * describes significance with p = 0.05 between control group and nanoparticle group at 2 Gy, determined by Mann–Whitney U-test; (g) UD-SCC-2; (h) UM-SCC-47.

Article Snippet: Doubling times of the (c) RPMI 2650 and (f) Cal33 cells at doses from 0 to 8 Gy. (g) Exempl ry growth cu ves of cell line (BEAS-2B) with and without 20 μg Fe/mL SPIONs and at 0 and 2 Gy over 120 h. (h) Doubling times of the cell lines BEAS-2B, CLS 354, Det 562, HSC-4, UD-SCC-2, and UM-SCC-47, each with and without SPIONs at doses of 0 and 2 Gy. (i) Significances between the different groups of (h); error bars indicate the standard deviation.

Techniques: Staining, Colony Assay, Control, MANN-WHITNEY

Journal: Nanomaterials (Basel, Switzerland)

Article Title: In Vitro Analysis of Superparamagnetic Iron Oxide Nanoparticles Coated with APTES as Possible Radiosensitizers for HNSCC Cells.

doi: 10.3390/nano13020330

Figure Lengend Snippet: Figure 6. Analysis of the cytostatic effect of SPIONs at 20 µg Fe/mL via growth curves through 24-well microscopy. Growth curves of (a) RPMI 2650 and (d) Cal33 cells from 0 to 8 Gy, each with and without SPIONs over 120 h; for reasons of clarity, they are only depicted at 0, 2, and 6 Gy; dashed lines show nanoparticle group. Exemplary presentation of all doses of (b) RPMI 2650 cells at 90 h and (e) Cal33 cells at 105 h (end of exponential growth). Doubling times of the (c) RPMI 2650 and (f) Cal33 cells at doses from 0 to 8 Gy. (g) Exemplary growth curves of cell line (BEAS-2B) with and without 20 µg Fe/mL SPIONs and at 0 and 2 Gy over 120 h. (h) Doubling times of the cell lines BEAS-2B, CLS 354, Det 562, HSC-4, UD-SCC-2, and UM-SCC-47, each with and without SPIONs at doses of 0 and 2 Gy. (i) Significances between the different groups of (h); error bars indicate the standard deviation. * equals significance between control group and SPION group at respective radiation dose, determined by Mann–Whitney U-test with p = 0.05.

Article Snippet: Doubling times of the (c) RPMI 2650 and (f) Cal33 cells at doses from 0 to 8 Gy. (g) Exempl ry growth cu ves of cell line (BEAS-2B) with and without 20 μg Fe/mL SPIONs and at 0 and 2 Gy over 120 h. (h) Doubling times of the cell lines BEAS-2B, CLS 354, Det 562, HSC-4, UD-SCC-2, and UM-SCC-47, each with and without SPIONs at doses of 0 and 2 Gy. (i) Significances between the different groups of (h); error bars indicate the standard deviation.

Techniques: Microscopy, Standard Deviation, Control, MANN-WHITNEY

Journal: Advanced Science

Article Title: Super‐Enhancer Driven LIF/LIFR‐STAT3‐SOX2 Regulatory Feedback Loop Promotes Cancer Stemness in Head and Neck Squamous Cell Carcinoma

doi: 10.1002/advs.202404476

Figure Lengend Snippet: LIF‐SE drives aberrant transcriptional activation of LIF in HNSCC. A. Genomic tracks plots displaying the H3K27ac HiChIP loop, H3K27ac ChIP‐seq peaks, and ATAC‐seq peaks of Cal27 at the LIF‐SE region. LIF‐SE contained five cis‐regulatory elements (referred to as E1, E2, E3, E4 and LIF‐Promoter). Cal27 H3K27ac HiChIP data analysis revealed the connections between enhancer fragments and LIF promoter within LIF‐SE. Our ChIP‐qPCR primers design was illustrated below; B) ABC tracks illustrated that E1‐E4 all have high predicted contact (ABC score) with LIF promoter; C) Heatmap plot displayed the average mRNA changes as measured by qRT‐PCR after indicated epigenetic chemicals treatments in Cal27 or Fadu cells; D) LIF pre‐mRNA and mRNA were measured by qRT‐PCR in Cal27 and Fadu cells treated with JQ1 (1 µ m ) or NEO2734 (500 n) at indicated time points (0, 1, 3, 6, 12, 24 h); E) ATAC‐seq and RNA‐seq tracks displayed the changes in chromatin accessibility and LIF transcriptional levels after JQ1 treatment in Cal33 cells; F) ATAC‐seq tracks showed the chromatin accessibility of LIF‐SE in HNSCC samples from the TCGA‐HNSC dataset; G) E1‐E4 core enhancer regions were cloned upstream of the luciferase promoter and further used for luciferase reporter assays, respectively; H) The binding changes of BRD4 and EP300 on E1‐E4 and LIF‐promoter after JQ1 exposure (1 µ m , 12 h) or NEO2734 (500 n m , 12 h) were measured by ChIP‐qPCR; I) Schematic diagram showing the procedure of E1‐E4 repression by dCas9‐KRAB‐MeCP2 system; J) LIF mRNA levels in Cal27‐dCas9 and Fadu‐dCas9 cells with repressed LIF enhancer activity were assessed by qRT‐PCR (left panel) and LIF titers in supernatants were measured by ELISA (right panel). Student's t ‐test. * p < 0.05, ** p < 0.01.

Article Snippet: A panel of cell lines, including Cal27, Cal33, Fadu and HEK293T, sourced from China Center for Type Culture Collection (CCTCC, Shanghai, China) was underwent authentication by short tandem repeat (STR) profiling and routinely tested negative for mycoplasma contamination.

Techniques: Activation Assay, HiChIP, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR, RNA Sequencing, Clone Assay, Luciferase, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Super‐Enhancer Driven LIF/LIFR‐STAT3‐SOX2 Regulatory Feedback Loop Promotes Cancer Stemness in Head and Neck Squamous Cell Carcinoma

doi: 10.1002/advs.202404476

Figure Lengend Snippet: SOX2/SMAD3 facilitates LIF transcription and CSCs stemness via binding with LIF‐SE in HNSCC. A. Representative images of primary tumorsphere formed by indicated enhancer‐repressed tumor cells; B) De novo motif analyses of individual constituent enhancers in LIF‐SE by HOMER algorithm; C) The integrated footprint profiles of SOX2 and SMAD3 derived from ATAC‐seq datasets across 4 cell lines were depicted (upper panel). The SOX2 and SMAD3 binding in selected SCC cell lines were displayed (lower panel); D,E) Endogenous LIF protein changes were measured in Cal27 and Fadu cells upon rhTGFβ−1 exposure (20 ng mL −1 , 48 h) or SOX2/SMAD3 knockdown; F) LIF pre‐mRNA and mRNA were measured by qRT‐PCR in Cal27 and Fadu cells treated with rhTGFβ−1 (20 ng mL −1 ) at indicated time points (0, 1, 3, 6 h); G) Increased luciferase activities of E1 reporter were observed in HEK293T cells transfected with increased dosages of SOX2 plasmid, while increased luciferase activities of E1, 2, 4 reporters were observed after exogenous SMAD3 overexpression; H) The SOX2 and SMAD3 binding at E1‐E4 and LIF promoter in Cal27, Cal33, and Fadu cells were detected by ChIP‐qPCR; I,J) SOX2 or SMAD3 silencing reduced CSC frequencies and downregulated LIF protein expression in Fadu xenografts; K) Chromatin accessibility in monolayer or 3D sphere cultured Cal27 cells was measured by ATAC‐seq, respectively; L) The relative binding of H3K27ac, SOX2, and SMAD3 in Cal27 and Fadu monolayer or tumorsphere cells was assessed by ChIP‐qPCR; M) Representative mIHC images showed the LIF and SOX2 co‐staining within CK + tumor cells in HNSCC samples. Scale bar: 100 µm. Student's t ‐test. # p ≥ 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: A panel of cell lines, including Cal27, Cal33, Fadu and HEK293T, sourced from China Center for Type Culture Collection (CCTCC, Shanghai, China) was underwent authentication by short tandem repeat (STR) profiling and routinely tested negative for mycoplasma contamination.

Techniques: Binding Assay, Derivative Assay, Knockdown, Quantitative RT-PCR, Luciferase, Transfection, Plasmid Preparation, Over Expression, ChIP-qPCR, Expressing, Cell Culture, Staining

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

doi: 10.21053/ceo.2022.01480

Figure Lengend Snippet: The hub genes in the intersection based on a Venn analysis

Article Snippet: The cell proliferation assays showed that the cell viability ( ) and colony formation capability ( ) were significantly weakened in the SERPINE1 -knockdown cells (sh- SERPINE1 -CAL27 and sh- SERPINE1 -SAS) relative to the controls ( P <0.05).

Techniques:

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

doi: 10.21053/ceo.2022.01480

Figure Lengend Snippet: (A) The expression of SERPINE1 in pan-cancer based on The Cancer Genome Atlas (TCGA) database. SERPINE1 expression was significantly higher in head and neck cancer (HNC) tissues than in normal tissues ( P <0.001). (B, C) The survival curves showed that HNC patients with high SERPINE1 expression had a shorter overall survival time than those with low SERPINE1 expression ( P <0.05). (B) TCGA. (C) GSE65858. (D) Multivariate Cox regression analysis with different variables indicated that SERPINE1 was an independent prognostic factor for HNC ( P <0.05). (E) TNMplot presented that high expression of SERPINE1 was associated with metastasis ( P <0.05). (F) The predicted targets of SERPINE1 . The interaction plot showed 57 target genes that may be closely related to SERPINE1 . (G, H) Gene Ontology (GO; G) and Kyoto Encyclopedia of Genes and Genomes (KEGG; H) enrichment analyses of the 57 genes. TPM, transcripts per million; HR, hazard ratio; CI, confidence interval; pT, pathological tumor; pN, pathological N; pTNM, pathological tumor-node-metastasis; TGF, transforming growth factor. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The cell proliferation assays showed that the cell viability ( ) and colony formation capability ( ) were significantly weakened in the SERPINE1 -knockdown cells (sh- SERPINE1 -CAL27 and sh- SERPINE1 -SAS) relative to the controls ( P <0.05).

Techniques: Expressing

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

doi: 10.21053/ceo.2022.01480

Figure Lengend Snippet: Associations between SERPINE1 expression and clinicopathological factors in oral cancer (TCGA)

Article Snippet: The cell proliferation assays showed that the cell viability ( ) and colony formation capability ( ) were significantly weakened in the SERPINE1 -knockdown cells (sh- SERPINE1 -CAL27 and sh- SERPINE1 -SAS) relative to the controls ( P <0.05).

Techniques: Expressing

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

doi: 10.21053/ceo.2022.01480

Figure Lengend Snippet: (A) The association between SERPINE1 expression and tumor purity, as well as the infiltration levels of several immune cells (TIMER algorithm). (B) Analysis of the relationship between the expression of SERPINE1 and the infiltration levels of 22 types of immune cells by the Cibersort algorithm. The darker color indicates a higher correlation ( * P <0.05). (C) The correlation of SERPINE1 expression with malignant phenotypes in head and neck cancer tissues. Scatter plots showed positive correlations between SERPINE1 expression and malignant phenotypes, such as (C1) metastasis, (C2) hypoxia, (C3) the epithelial-mesenchymal transition, and (C4) angiogenesis. (D) The relationship between SERPINE1 expression and the drug sensitivity of cancer cells. Red represents a positive correlation, while blue stands for a negative correlation. TPM, transcripts per million; TCGA, The Cancer Genome Atlas; HNSC, Head and Neck squamous cell carcinoma; NK, natural killer; EMT, epithelial-mesenchymal transition; GDSC, Genomics of Drug Sensitivity in Cancer; mRNA, messenger RNA; FDR, false discovery rate.

Article Snippet: The cell proliferation assays showed that the cell viability ( ) and colony formation capability ( ) were significantly weakened in the SERPINE1 -knockdown cells (sh- SERPINE1 -CAL27 and sh- SERPINE1 -SAS) relative to the controls ( P <0.05).

Techniques: Expressing

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

doi: 10.21053/ceo.2022.01480

Figure Lengend Snippet: (A) The messenger RNA (mRNA) expression of SERPINE1 was higher in nicotine-treated oral cells (DOK/NIC) and oral cancer cell lines (Cal27, SAS, HSC-3) than that in DOK cells, respectively. (B) The immunohistochemistry scores of SERPINE1 protein expression in oral cancer tissues were markedly higher than in the normal controls. (C) The expression scores for SERPINE1 protein were higher in cancer samples with lymph node metastasis (LNM) than in those without LNM. (D) The scores of SERPINE1 expression were higher in the samples with high pathological stages than in those with low stages. No associations were presented concerning age (E), sex (F), and Tstage (G). IHC, immunohistochemistry; NS, not significant ( P >0.05). * P <0.05.

Article Snippet: The cell proliferation assays showed that the cell viability ( ) and colony formation capability ( ) were significantly weakened in the SERPINE1 -knockdown cells (sh- SERPINE1 -CAL27 and sh- SERPINE1 -SAS) relative to the controls ( P <0.05).

Techniques: Expressing, Immunohistochemistry

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

doi: 10.21053/ceo.2022.01480

Figure Lengend Snippet: (A) The mRNA expression of SERPINE1 was significantly downregulated in the SERPINE1 -silenced oral cancer cells (sh- SERPINE1 -Cal27 or sh- SERPINE1 -SAS) compared with that of the control cells (sh-NC-Cal27 and sh-NC-SAS). (B) The trend of SERPINE1 protein expression was in line with that of mRNA expression. (C) The cell proliferation abilities of the SERPINE1 -silenced cancer cells were significantly lower than those of the control cells. (D) The number of colonies formed in the SERPINE1 -silenced cells was significantly lower than that in the control cells. (E) The invasive abilities in the SERPINE1 -silenced cells were significantly inhibited compared with those in the control cells. (F) The administration of Bleomycin or docetaxel resulted in a significant decrease in cell viability in SERPINE1 -silenced cells compared with the control cells. mRNA, messenger RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; OD, optical density. * P <0.05.

Article Snippet: The cell proliferation assays showed that the cell viability ( ) and colony formation capability ( ) were significantly weakened in the SERPINE1 -knockdown cells (sh- SERPINE1 -CAL27 and sh- SERPINE1 -SAS) relative to the controls ( P <0.05).

Techniques: Expressing, Control, Negative Control