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: Nature Communications

Article Title: Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch

doi: 10.1038/ncomms10215

Figure Lengend Snippet: ( a ) One day after the knockdown (siRM) of endogenous RepoMan in HeLa Flp-in T-Rex cells, siRNA-resistant EGFP-RepoMan fusions were induced for 24 h with doxycycline (Dox). Endogenous RepoMan and the EGFP-fusions were visualized by immunoblotting with pan-RepoMan antibodies. ( b ) Same as in a but the cells were subjected to time-lapse imaging by confocal microscopy after release from a 6 h-Monastrol arrest. The panel shows cells at different time points after the initiation of anaphase. DNA was stained with Hoechst 33342. Scale bars, 5 μm. ( c ) Quantification of the chromosome targeting (EGFP/DNA ratio) of the EGFP-RepoMan fusions as shown in ( b ). The means±s.e.m. of at least 11 cells are shown in each condition. Data were normalized to the maximal ratio of EGFP/DNA in each condition. ( d ) Live imaging of Nocodazole-arrested U2OS cells after expression of EGFP-tagged RepoMan or its phosphatase/histone-binding mutants. The figure shows the localization of DNA and the EGFP fusions. Scale bars, 5 μm. ( e ) Quantification of the ratio of EGFP signal on chromosome versus cytoplasm, as shown in d . The means±s.e.m. of at least 12 cells were analysed in each condition. *** P <0.001 in paired t -test, as compared with WT.

Article Snippet: Calyculin A (EI-192, Biomol), Roscovitine (R-1234, LC Laboratories), Hesperadin (S1529, Selleckchem), Monastrol (orb146117, Biorbyt) and Microcystin-LR (10007188, Cayman) were purchased.

Techniques: Knockdown, Western Blot, Imaging, Confocal Microscopy, Staining, Expressing, Binding Assay

Journal: Nature Communications

Article Title: Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch

doi: 10.1038/ncomms10215

Figure Lengend Snippet: ( a ) HeLa Flp-in T-Rex cells were induced to express siRNA-resistant EGFP-tagged RepoMan-WT or RepoMan-3A following the knockdown of endogenous RepoMan. Monastrol-arrested mitotic cells were fixed before staining. Scale bars, 5 μm. ( b ) Quantification of the H3T3ph/DNA ratio in a . The data represent means±s.e.m. for three independent experiments (≥11 cells per condition in each experiment). ( c ) Cells were treated as in a but stained for Aurora B, Aurora-B -T232ph and DNA. Scale bars, 5 μm. ( d ) Quantification of the T232ph/Aurora-B ratio in c . The data represent means±s.e.m. for three independent experiments (≥11 cells per condition in each experiment) with paired t -test, as compared with siCtrl. ( e ) Cells were treated as in a but stained for H3S10ph, Aurora-B and DNA. Scale bars, 5 μm. ( f ) Quantification of the H3S10ph/DNA ratio in e . The data represent means±s.e.m. for three independent experiments (≥10 cells per condition in each experiment). ( g ) Cell-treatment scheme for chromosome alignment assays of HeLa Flp-in T-Rex cells, before and after induction of EGFP-tagged RepoMan-WT or RepoMan-3A, and following the knockdown of endogenous RepoMan. ( h ) In cells treated as explained in g , the percentage of cells with a well-aligned metaphase plate or unaligned chromosomes were plotted as mean percentages±s.e.m. for three independent experiments (≥121 cells per condition in each experiment). ( i ) Cells were treated as in a but stained for Mad2, ACA and DNA. ( j ) Quantification of relative signal of centromeric Mad2 in i . Data were normalized to the indicated ratio as means±s.e.m. for three independent experiments (≥8 centromeres per cell, ≥10 cells per condition in each experiment). ( k ) Traps of EGFP-fusions with RepoMan-WT or RepoMan-RATA from non-synchronized HEK293T cells were used as a phosphatase source to dephosphorylate recombinant GST-Aurora B/INCENP. The figure shows different time points of incubation with the traps analysed by immunoblotting. * P <0.05; ** P <0.01; *** P <0.001, with paired t -test as compared with siCtrl.

Article Snippet: Calyculin A (EI-192, Biomol), Roscovitine (R-1234, LC Laboratories), Hesperadin (S1529, Selleckchem), Monastrol (orb146117, Biorbyt) and Microcystin-LR (10007188, Cayman) were purchased.

Techniques: Knockdown, Staining, Recombinant, Incubation, Western Blot