Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: Combinative treatment of β-elemene and cetuximab was sensitive to KRAS mutant CRC cells. (A) The sensitivity of KRAS mutant and wild-type colorectal cancer cells to cetuximab treatment (25 µg/ml) for 24 h was detected by CCK-8 assay. The mean ± s.d. is shown. ** P < 0.01. (B) The inhibitory effects and cytotoxicity of co-treatment with β-elemene (125 µg/ml) and cetuximab (25 µg/ml) in KRAS mutant CRC cells was determined after the treatment for 24 h. (C) Representative cell morphological changes are detected by light microscopy. Scale bar = 100 μm. (D) Representative results of annexin V-FITC/PI staining and quantitative analysis after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. The mean ± s.d. is shown. ** P < 0.01. (E) Representative results of cell cycle and quantitative analysis after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. (F) The colony-formation assay was performed and colony numbers are shown (β-elemene 125 µg/ml, cetuximab 25 µg/ml). The mean ± s.d. is shown. ** P < 0.01.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Mutagenesis, CCK-8 Assay, Light Microscopy, Staining, Colony Assay

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The effect of co-treatment with β-elemene and cetuximab on several ferroptotic events in KRAS mutant CRC cells. (A) The effect of cetuximab and β-elemene in combination with other cell death inhibitors on the cell viability of KRAS mutant HCT116 and Lovo cells after the treatment for 24 h. The mean ± s.d. is shown. (B) The cellular ROS level after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h was analyzed by a flow cytometer, ** P < 0.01. (C) Intracellular GSH level in KRAS mutant HCT116 and Lovo cells after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h was detected, ** P < 0.01. (D) Intracellular MDA levels in KRAS mutant HCT116 and Lovo cells after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h was detected, ** P < 0.01.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Mutagenesis, Flow Cytometry

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The iron ion level and mitochondria staining were detected. (A) The chelatable iron was determined using the fluorescent indicator Phen Green SK (green) after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. Scale bar = 100 µm. (B) The Mitochondria morphology was assessed with Mito-Tracker Green after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. Scale bar = 50 µm.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Staining

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The effect of co-treatment with β-elemene and cetuximab on ferroptosis-related proteins in KRAS mutant CRC cells. (A) The expression of positive regulatory proteins for ferroptosis (HO-1 and transferrin) and the negative regulatory proteins for ferroptosis (GPX4, SLC7A11, FTH1, glutaminase, and SLC40A1) were detected by western blotting after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. (B) HCT116 and Lovo cells were treated with cetuximab (25 µg/ml) and β-elemene (125 µg/ml) with or without ferroptosis inhibitors for 24 h and cell viability was assayed. The mean ± s.d. is shown. ** P < 0.01.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Mutagenesis, Expressing, Western Blot

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: Combinative treatment of β-elemene and cetuximab suppressed the migration of KRAS mutant CRC cells by inhibiting EMT. (A) Representative results of wound healing after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml, DFO 20 nM) for 24 h. The mean ± s.d. is shown. ** P < 0.01. (B) Transwell invasion assay was performed by the 24-transwell system and quantitative analysis. The pictures were taken 24 h after seeding (original magnification: × 100). The mean ± s.d. is shown. ** P < 0.01. (C) The expression of several key EMT markers Vimentin, E-Cadherin, N-Cadherin, Slug, Snail and MMP-9 were detected after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h by western blotting.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Migration, Mutagenesis, Transwell Invasion Assay, Expressing, Western Blot

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The antitumor efficacy of co-treatment with β-elemene and cetuximab in vivo . (A) The scheme of tumor inoculation and systemic injection. (B) Bioluminescent imaging for HCT116-luc orthotopic xenograft colon tumors at different time points post treatment (β-elemene 50 mg/kg, cetuximab 50 mg/kg) and representative image of metastatic lymph nodes. (C) Fold change in average radiance per mouse at experimental endpoint (day 18) was analyzed for each treatment group. Data are expressed as the mean ± s.d. (D) The survival curves of mice in each group were assessed.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: In Vivo, Injection, Imaging

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: A) Overview of experimental design. B) Protein stain for TP63 (green), EGFR (pink), F3 (white) and DAPI (blue) in organoids treated for 3 days of DSA followed by 7 days of DSI. Scale bar represents 50 μm. C) EGFR/F3 and control FACS plots for 1 representative biological replicate (n=3 biological replicates). D-E) Protein staining for TP63 (red, nuclear) and quantification of TP63+ cells/total cells on sorted cells spun onto a glass slide using a Cytospin. Error bars represent the mean +/− the standard error of the mean. N=3 biological replicates, shown as 3 separate colors on the plot. F) Quantification of the percentage of cells in each organoid containing positive protein staining for canonical cell type markers in unsorted organoids versus organoids that were grown from isolated EGFR+/F3+ cells. N=3 biological replicates per group and n=3 technical replicates (individual organoids) per biological replicate. A total of 198 cells were counted for the EGFR-/F3- group, 110 cells counted for EGFR+/F3- group, 38 cells counted for the EGFR-/F3+ group, and 88 cells counted for the EGFR+/F3+ group. G) Protein staining of organoids derived from EGFR/F3 sorted cells for secretory marker SCGB1A1 (pink), multiciliated markers AcTUB+ and FOXJ1+ (white), epithelial marker KRT8 (green), basal cell marker TP63 (pink), goblet cell marker MUC5AC (white) and DAPI (blue). Data shown from a single biological replicate and is representative of N=3 biological replicates. Scale bars represents 50 μm. H)GFP (green), RFP (red) and brightfield images of whole organoids 11 days after re-plating and mixing EGFR/F3 sorted cells from GFP and RFP expressing groups. N=1 biological replicate with n=6 technical replicates (wells of mixed organoids). Data shown from a single experiment and is representative of n=3 experiments. Scale bar represents 200 μm (top row) and 100 μm (bottom row).

Article Snippet: Human anti-EGFR-APC (FACS) , Milteny , 130-110-587.

Techniques: Staining, Control, Isolation, Derivative Assay, Marker, Expressing

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human anti-EGFR-APC (FACS) , Milteny , 130-110-587.

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software

Journal: Frontiers in Oncology

Article Title: Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients

doi: 10.3389/fonc.2020.572895

Figure Lengend Snippet: Assessment of different blood collection tubes for EGFR assays on cfDNA and CTC DNA after 24 and 48 h of blood storage. (A) Overall performance of the different BCTs. LBgard is identified as the preferred BCT as opposed to EDTA, which performance significantly worsens over the storage duration. (B) Initial blood cell viability in the plasma-depleted blood sample, before VTX-1 processing. (C) Presence of numerous DAPI + debris, i.e., nucleus from dead cells, in the VTX-1 output from EDTA BCTs after 48 h. (D) cfDNA yield from the plasma workflow. (E) Cell DNA yield from the cell workflow after VTX-1 processing. (F) EGFR mutation detection for cfDNA and CTC DNA, at Day 0, 1, and 2. All pictures are original.

Article Snippet: After a centrifugation (600 g , 1 min, RT) and aspiration of the supernatant, cells were fixed with 2% PFA (Electron Microscopy Sciences #157-4) for 10 min, permeabilized with 0.2% volume/volume Triton X-100 (Research Products International Corp) and 5% Goat Serum (Invitrogen) for 7 min, blocked with 10% Goat Serum for 30 min, and immunostained. (i) For the experiments assessing the different blood collection tubes , immunostaining was performed using antibodies directed against cytokeratins (CK) (FITC, Clone CAM 5.2, BD Biosciences #347653; Clone CK3-6H5 Miltenyi Biotec #130080101), against CD45 (PE, Clone HI30, BD Pharmingen #555483) and counterstained with DAPI (Molecular Probes #D3571). (ii) For all EGFR spiking experiments and patient samples , cells were stained with anti-CK FITC (Clone CAM 5.2, BD Biosciences, #347653; Clone CK3-6H5, MACS Miltenyi, #130-080-101; Clone AE1/AE3, eBioscience, #53-9003-82), anti-Vimentin AF647 (Clone V9, Abcam, #195878), anti-N-Cadherin AF647 (Clone EPR1791-4, Abcam, #195186) and anti-CD45 PE (Clone HI30, BD Biosciences, #555483) and counterstained with DAPI.

Techniques: Clinical Proteomics, Mutagenesis