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