Journal: Scientific Reports

Article Title: Exceptionally selective voltage-sensor trapping of Na V 1.5 channels by Mg-protoporphyrin impairs cancer cell migration

doi: 10.1038/s41598-026-37492-0

Figure Lengend Snippet: MgPpIX slows down the migration of cancer cells expressing SCN5A. ( A ) Representative images of MDA-MB-231 cell cultures right after introducing a scratch gap (0 h) and 4 h later. After scratch introduction, the cells were kept in control medium with DMSO (Ctrl) or 100 nM of the indicated MePpIX, dissolved in DMSO. The cells were kept in the dark to prevent phototoxicity induced by the MePpIXs. Continuous black lines indicate the automatically detected scratch borders. Scale bar: 400 µm. ( B ) Mean gap-closure migration of MDA-MB-231 cells (relative to the migration under control conditions, i.e., in the presence of the vehicle DMSO) in the presence of 100 nM of PpIX or the indicated MePpIX variants. C Representative confocal images of DAPI-stained MDA-MB-231 cell nuclei detected on the trans side in a transwell migration assay with an 8 µm pore diameter and a chemoattraction gradient from 0 (insert) to 2% FBS (wells); background in white. The panels on the right show superpositions of the DAPI staining (blue) with transmission images. Cells were kept overnight under control conditions or in the presence of 100 nM of the indicated MePpIX. Images were taken through a 10 × objective with a 3 × zoom factor. Cell counting was performed based on the DAPI staining using ImageJ software. Scale bar: 400 µm. ( D ) Mean number of migrated cells from experiments as shown in ( C ). E Mean migration in a wound-healing assay of MDA-MB-231 cells over 4 h in the presence of the indicated concentrations of MgPpIX, TTX, and PpIX. ( F ) Mean normalized current traces obtained from MDA-MB-231 cells under voltage-clamp conditions with step depolarizations to − 30 mV without (black) and with the application of 100 nM MgPpIX. The currents on the right followed an episode of 250 ms at 40 mV to highlight the reverse use-dependence of these Na V channels. ( G ) Mean relative migration in a wound-healing assay (as in B and E ) but for T-47D cells. ( H ) As in ( G ) but for the colon carcinoma cell lines SW-480 and SW-48. ( I ) Mean current density, based on the peak currents at − 30 mV, for HEK293T cells and the indicated breast cancer (MDA-MB-231, T-47D) and colon carcinoma cells (SW-480, SW-48). Data bars are means ± sem with n in parentheses; in ( B ), ( D ), ( E ), ( G ), and ( H ), the number of independent cell batches is given.

Article Snippet: The breast cancer cell lines MDA-MB-231 (DSMZ) and T-47D (American Type Culture Collection, ATCC) were cultured in RPMI 1640 medium (Sigma, R7388), while the colorectal cancer cell lines SW-480 (DSMZ) and SW-48 (ATCC) were maintained in DMEM.

Techniques: Migration, Expressing, Control, Staining, Transwell Migration Assay, Transmission Assay, Cell Counting, Software, Wound Healing Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: CRISPR-Cas9 gene editing strengthens cuproptosis/chemodynamic/ferroptosis synergistic cancer therapy

doi: 10.1016/j.apsb.2024.05.029

Figure Lengend Snippet: In vitro distribution and tumor-combatting effect of RNP@Cu 2 O@SPF. (A) The viability of cancer and normal cells treated with RNP@Cu 2 O@SPF for 24 h in the dark ( n = 6). (B) CLSM images of SW480 cells incubated with RNP@Cu 2 O@SPF or RNP@Cu 2 O@S (FITC-labelled RNP, RITC-labelled silica). Scale bar, 20 μm. (C) FCM analysis showcasing transfection rates in SW480 cells after 4 h of immersion with PBS, RNP@Cu 2 O@S, or RNP@Cu 2 O@SPF. WB assay (D) and fluorescence imaging (E) of ATP7A expression in SW480 cells treated with RNP@Cu 2 O@S or RNP@Cu 2 O@SPF. Scale bar, 100 μm. (F) Relative copper contents in SW480 cells after a 24 h treatment with Cu 2 O@SPF or RNP@Cu 2 O@SPF ( n = 6). (G) Fluorescence microscopy images of ROS generation in SW480 cells treated with PBS, Cu 2 O@SPF(40 μg/mL), RNP@Cu 2 O@SPF (40 μg/mL), and RNP@Cu 2 O@SPF (80 μg/mL) for 4 h and stained with DCFH-DA to indicate the ROS generation. Scale bar, 50 μm. (H) GSH/GSSG ratio in SW480 cells treated with different concentrations of RNP@Cu 2 O@SPF (25, 50, 100 μg/mL) for 6 h ( n = 6). The data are shown as mean ± SD. ns, no significant; ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The Cell Copper (Cu) Colorimetric Assay Kit (Elabscience Biotechnology, Wuhan, China) was selected to detect the copper ion content in SW480 cells, and the BCA Protein Colorimetric Assay Kit (Elabscience Biotechnology, Wuhan, China) was also used to determine the total protein concentration.

Techniques: In Vitro, Incubation, Transfection, Fluorescence, Imaging, Expressing, Microscopy, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: CRISPR-Cas9 gene editing strengthens cuproptosis/chemodynamic/ferroptosis synergistic cancer therapy

doi: 10.1016/j.apsb.2024.05.029

Figure Lengend Snippet: Anti-tumor mechanisms studies of RNP@Cu 2 O@SPF. (A) The schematic diagram of cell death mechanisms of RNP@Cu 2 O@SPF nanomedicine-mediated synergetic therapy. (B) FCM analysis with Annexin V-FITC/PI dual labels on SW480 cells after 24 h of incubation with different nanoparticles. (C) The percentages of cells undergoing apoptosis (%) in different groups ( n = 3). (D) Viability of SW480 cells grown in media containing either glucose or galactose treated with RNP@Cu 2 O@SPF (ratio 1:1) ( n = 6). (E) Viability of SW480 cells pretreated with 0.1 mmol/L rotenone (Rot), 0.1 mmol/L antimycin A (anti-A), or 1 mmol/L FCCP and then treated with RNP@Cu 2 O@SPF ( n = 6). (F) Viability of SW480 cells post-treated with Antimycin A or UK5099 under different conditions (incubated with various nanoparticles) ( n = 6). (G) Viability of SW480 cells treated with tetrathiomolybdate in different concentrations of RNP@Cu 2 O@SPF (50, 100 μg/mL) ( n = 6). (H) Western blot analysis of DLAT-oligomers, DLAT, and Tubulin expression levels in SW480 cells after treatment with different nanoparticles. (I) Western blot analysis of lipoylated proteins, FDX1, LIAS, ACO-2, SDHB9, and Tubulin expression levels in SW480 cells after treatment with different nanoparticles. (J) Fluorescence depiction of GPX4 levels in SW480 cells post RNP@Cu 2 O@SPF treatment at different dosages (25, 50, and 100 μg/mL) for 24 h. Scale bar, 25 μm. (K) LPO contents in SW480 cells treated with RNP@Cu 2 O@SPF at different concentrations (25, 50, and 100 μg/mL) for 24 h ( n = 6). (L) Survival rates of SW480 cells after 24 h of incubation with solutions of PBS, RNP@Cu 2 O@SPF, PBS+DFO, or RNP@Cu 2 O@SPF+DFO ( n = 6). (M) WB assay of GPX4, ACSL4, and Tubulin expression in SW480 cells treated with RNP@Cu 2 O@SPF at varied concentrations (25, 50, and 100 μg/mL) for 24 h. The data are shown as mean ± SD. ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The Cell Copper (Cu) Colorimetric Assay Kit (Elabscience Biotechnology, Wuhan, China) was selected to detect the copper ion content in SW480 cells, and the BCA Protein Colorimetric Assay Kit (Elabscience Biotechnology, Wuhan, China) was also used to determine the total protein concentration.

Techniques: Incubation, Western Blot, Expressing, Fluorescence

Journal: Oncogene

Article Title: Overexpression of the thymosin beta-4 gene is associated with increased invasion of SW480 colon carcinoma cells and the distant metastasis of human colorectal carcinoma.

doi: 10.1038/sj.onc.1207888

Figure Lengend Snippet: Figure 1 Tb-4 overexpression promotes the in vivo growth of SW480 human colon carcinoma cells. A tumor xenograft growth assay was performed using stable transfectants derived from the SW480 cells established previously by us (Wang et al., 2003) including 480S1 & 480S2 (Tb-4-overexpressing clones), 480BK (vector control), and the parental cells. After being trypsinized and washed once in PBS, cells were then resuspended in PBS at a concentration of 2 106 cells/ml. A total of 1 106 cells were injected subcutaneously onto the back of 8-week-old athymic nude mice (National Laboratory Animal Center, Nankang, Taiwan), and the size of the growing tumors was determined by measuring their length and width with calipers every week (a). The weight of the tumors was measured at the 7th week after the mice were killed (b). Data are means7s.d. from five independent determinations. *Po0.05 when compared with the parental and vector-transfected SW480 cells by one-way ANOVA with repeated measures

Article Snippet: Interestingly, decreased Fas expression Figure 2 Tb-4 overexpression promotes both the in vitro and in vivo invasion of SW480 cells. (a) A measure of 50 mg of total lysates prepared from the SW480, 480BK, 480S1, and 480S2 cells were separated on a 10% SDS–polyacrylamide gel and processed for immunoblotting using an anti-MMP-7 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA).

Techniques: Over Expression, In Vivo, Growth Assay, Derivative Assay, Clone Assay, Plasmid Preparation, Control, Concentration Assay, Injection, Transfection

Journal: Oncogene

Article Title: Overexpression of the thymosin beta-4 gene is associated with increased invasion of SW480 colon carcinoma cells and the distant metastasis of human colorectal carcinoma.

doi: 10.1038/sj.onc.1207888

Figure Lengend Snippet: Figure 2 Tb-4 overexpression promotes both the in vitro and in vivo invasion of SW480 cells. (a) A measure of 50 mg of total lysates prepared from the SW480, 480BK, 480S1, and 480S2 cells were separated on a 10% SDS–polyacrylamide gel and processed for immunoblotting using an anti-MMP-7 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA). Similar blot was reprobed with an anti-b-tubulin antibody (BD, San Diego, CA, USA) to show equal loading of each sample. (b) The enzymatic activity of the matrix metalloproteinases (MMPs) in different cell lines was determined by proteolytic cleavage of a fluorogenic substrate Ac-Pro-Leu-[2- mercapto-4-methyl- pentanoyl]-Leu-Gly-OC2H5 (Biomol, Butler Pike, PA, USA) using total cell lysates as enzyme sources. The assays were carried out with 50 mg cell lysates in a buffer (50 mM HEPES, 10 mM CaCl2, 0.05% Brij-35, pH 7.0) at 301C and fluorescence was measured by a spectrofluorometer using excitation and emission wavelengths at 340 and 440 nm, respectively. The initial rates were calculated from the changes in fluorescence as described previously (Netzel-Arnett et al., 1991) using substrate concentrations of 10, 20, 30, 40, 60, 80, 100, 150 mM. Each substrate concentration was assayed three times and the kinetic curves were plotted. (c) In vitro invasion of these cells was assessed by examining their abilities to invade through the transwells coated with basement membrane Matrigel as previously described (Albini et al., 1987). Polycarbonate filters (12 mm pore size) in the Boyden chamber (Becton Dickinson & Co., Oxford, CA, USA) were coated with 100 ml (0.95 mg of protein/ml) of Matrigel matrix (BD Bioscience, Benford, MA, USA). Single cell suspensions obtained by trypsinization were placed into the upper chamber in L-15 medium (Life Technologies, Rockville, MD, USA) containing 10% fetal calf serum at a density of 105 cells/well. After incubation at 371C for 24 h, cells that did not penetrate the filter were wiped off by PBS-rinsed cotton swabs, and cells that had migrated to the lower surface of the filter were stained with Hoechst33258 and counted by a fluorescence microscope (Olympus, Tokyo, Japan). Values for invasion were expressed as the average number of migrated cells per microscopic field over six fields per assay from three independent experiments. Data are means7s.d. from three independent determinations. *Po0.05 when compared with the parental and vector-transfected SW480 cells by one-way ANOVA with repeated measures. Significant increases in adhesion and infiltration of the tumor cells derived from Tb-4 overexpressers to their adjacent skin (d) and muscle (e) tissues were found in nude mice. Tumor tissue collection from killed animals was performed in the Animal Resource Center of the National Yang-Ming University. Postmortem studies including tumor sectioning, H&E staining, and microscopic examination were performed by an experienced pathologist at Taipei Veterans General Hospital. * Photographs A, B, C, and D represent cell lines SW480, 480BK, 480S1, and 480S2, respectively, with a magnification of 400

Article Snippet: Interestingly, decreased Fas expression Figure 2 Tb-4 overexpression promotes both the in vitro and in vivo invasion of SW480 cells. (a) A measure of 50 mg of total lysates prepared from the SW480, 480BK, 480S1, and 480S2 cells were separated on a 10% SDS–polyacrylamide gel and processed for immunoblotting using an anti-MMP-7 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA).

Techniques: Over Expression, In Vitro, In Vivo, Western Blot, Activity Assay, Concentration Assay, Membrane, Pore Size, Incubation, Staining, Microscopy, Plasmid Preparation, Transfection, Derivative Assay

Journal: Oncogene

Article Title: Overexpression of the thymosin beta-4 gene is associated with increased invasion of SW480 colon carcinoma cells and the distant metastasis of human colorectal carcinoma.

doi: 10.1038/sj.onc.1207888

Figure Lengend Snippet: Figure 3 Tb-4 overexpression reduces the levels of Fas and the susceptibility to FasL-mediated apoptosis of SW480 cells. (a) Total lysates (50 mg) prepared from the SW480, 480BK, 480S1, and 480S2 cells were separated on a 10% SDS–polyacrylamide gel and processed for immunoblotting using an anti-Fas polyclonal anti- body (Santa Cruz). A similar blot was reprobed with an anti-b- tubulin antibody (BD, San Diego, CA, USA) to show equal loading of each sample. (b) For analysis of the viability of different cell lines after treatment with a Fas ligand, 1 104 cells from each line were seeded in triplicate in a 96-well plate with 0.2 ml of culture medium for 24 h. An anti-Fas IgM (Upstate, Lake Placid, NY, USA), serves as a Fas ligand, was then added in a concentration of 40 ng/ml and incubated for an additional 48 h. The medium was then replaced by 0.1 ml of fresh medium containing 10 ml of MTT (5 mg/ml in PBS) (Sigma, St Louis, MO, USA). After incubation at 371C for 4 h, 0.1 ml lysis buffer (20% SDS in 50% DMF) was added and the absorbance at 570 nm was measured using a Microplate Reader (Model 550, BIO-RAD). Data are means7s.d. from three independent determinations. *Po0.05 when compared with the parental and vector-transfected SW480 cells by one-way ANOVA with repeated measures

Article Snippet: Interestingly, decreased Fas expression Figure 2 Tb-4 overexpression promotes both the in vitro and in vivo invasion of SW480 cells. (a) A measure of 50 mg of total lysates prepared from the SW480, 480BK, 480S1, and 480S2 cells were separated on a 10% SDS–polyacrylamide gel and processed for immunoblotting using an anti-MMP-7 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA).

Techniques: Over Expression, Western Blot, Concentration Assay, Incubation, Lysis, Plasmid Preparation, Transfection

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: OA-induced ROS production is dependent on NOX4 expression in CRC cells. (A) OA-induced ROS levels were examined by flow cytometry analysis with DCFDA staining in SW480 cells treated with OA for various concentrations (i) or periods of time (ii), as indicated. Quantification of ROS levels are shown in columns. BG indicates background. (B-C) Real-time quantitative PCR analysis was performed for detecting NOX1-5 (N1-5), SOD1-2 (S1-2), DUOX1-2 (D1-2) mRNA levels in SW480 (i) or HT-29 (ii) cells treated with 200 µM OA for 16 h (B) or the indicated period of time (C). (D) ROS levels and NOX4 protein expression were examined by flow cytometry analysis with DCFDA staining and western blotting, respectively. SW480 (i) and HT-29 (ii) cells were transfected with 20 nM scrambled oligonucleotides (SC) or NOX4 siRNA (siNOX4 #1 or #2) for 24 h and then treated with 200 µM OA for 24 h. BG indicates background. The data are presented as the mean ± SEM. P -values were determined using a two-tailed Student's t -test. * P < 0.05; ** P < 0.01; *** P < 0.001 (n=3).

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: Expressing, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: NOX4 expression is essential for OA-induced invasion ability in CRC cells. (A-B) Effects of ROS, NOX4, and MMPs on OA-induced cell invasion were analyzed using transwell invasion assays. SW480 (i, iii) and HT-29 cells (ii) were treated with 5 mM NAC or 15 µM Vitamin E (A) or transfected with 20 nM NOX4, MMP-1, and MMP-9 siRNA or scrambled oligonucleotides (SC) (B) followed by treatment with 200 µM OA for 72 h. Invading cells were stained with crystal violet and imaged with a microscope, and then solubilized in 10% acetic acid for quantification of invasive levels as shown in columns. The absorbance was measured at a wavelength of 595 nm. The siRNA knockdown efficiency was examined using real-time quantitative PCR (ii). (C) Real-time quantitative PCR analysis of NOX4, MMP-1 , MMP-3 , MMP-9, Vimentin, E-cadherin, and ZEB-1 expression in SW480 cells was performed in cells transfected with 20 nM siNOX4 or SC siRNA followed by treatment with 200 µM OA for 16 h. The data are presented as the mean ± SEM. P -values were determined using a two-tailed Student's t -test. * P < 0.05; ** P < 0.01; *** P < 0.001 (n=3).

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: Expressing, Transfection, Staining, Microscopy, Knockdown, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: OA-induced ANGPTL4 regulates NOX4 transcriptional activity through the AP-1 binding site on the NOX4 promoter. (A) Dual-luciferase reporter assay was performed to analyze the activation of NOX4 promoter (4.7 kb length) in SW480 cells. Cells were treated with 200 µM OA for the indicated period of time (i). The level of remaining NOX4 mRNA was analyzed by real-time quantitative PCR in SW480 cells treated with or without 200 µM OA for 3 h followed by incubation with 4 μM actinomycin D (ActD) for the indicated period of time (ii). (B) Dual-luciferase reporter assay was performed in cells transfected with a series of 5'-truncated NOX4 promoters followed by treatment with 200 µM OA for 16 h. (C-D) Dual-luciferase reporter assay and ELISAs were performed in SW480 cells transfected with the wild-type (WT) NOX4 promoter or with AP-1 binding site mutation (AP-1m) (C), and 20 nM siANGPTL4 (siANG) (D) (i, iii) or expression vector of ANGPTL4 (flANG) (D) (ii, iv), followed by treatment with 200 µM OA for 16 h. Firefly luciferase activity was determined and normalized to Renilla luciferase activity. The data are presented as the mean ± SEM. P -values were determined using a two-tailed Student's t -test. *** P < 0.001 (n=3).

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: Activity Assay, Binding Assay, Luciferase, Reporter Assay, Activation Assay, Real-time Polymerase Chain Reaction, Incubation, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: OA-induced secretion of ANGPTL4 enhances invasion ability in cancer cells. (A) Semi-quantitative PCR analysis was performed to examine ANGPTL4 mRNA levels in SW480 cells treated with OA at various concentrations (i) or periods of time (ii), as indicated. ELISAs were performed to asses ANGPTL4 secretion levels (iii). (B) Invasion assays were performed using SW480 cells transfected with 20 nM ANGPTL4 siRNA (siANG#1 or #2) or scrambled oligonucleotides (SC) and then treated with 200 µM OA, 1 µg/ml anti-ANGPTL4 antibody (ANG Ab), and 100 ng/ml recombinant human ANGPTL4 (rh-ANG) for 72 h. Invading cells were stained with crystal violet and imaged under a microscope (i), and then solubilized with 10% acetic acid. The absorbance was measured at a wavelength of 595 nm (ii). ELISAs were performed to assess ANGPTL4 secretion in SW480 cells transfected with 20 nM siANGPTL4 or SC siRNA followed by treatment with 200 µM OA for 24 h (iii). (C) Real-time quantitative PCR analysis of MMP-1 , MMP-3 , MMP-9, Vimentin, E-cadherin, and ZEB-1 mRNA levels was performed in SW480 cells transfected with 20 nM siANGPTL4 or SC siRNA and then treated with 200 µM OA for 16 h (i). ELISAs were performed to assess ANGPTL4 secretion (ii). The data are presented as the mean ± SEM. P -values were determined using a two-tailed Student's t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. (n=3).

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Recombinant, Staining, Microscopy, Two Tailed Test

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: OA-induced NOX4 and ROS production are regulated by ANGPTL4 in CRC cells. (A-B) ROS levels were analyzed by flow cytometry analysis with DCFDA staining in SW480 cells transfected with 20 nM siANGPTL4 (siANG#1 and #2), SC siRNA, or expression vector of ANGPTL4 (flANG) and then treated with or without 200 µM OA and 5 mM NAC for 24 h. Quantification of ROS intensities is shown in columns (i). BG indicates background. ELISAs were performed to assess ANGPTL4 secretion in SW480 cells (ii). Vec indicates empty vector. (C-E) Real-time quantitative PCR and immunoblotting analyses for NOX4 and ANGPTL4 mRNA and protein levels, respectively were performed in SW480 cells transfected with 20 nM siANGPTL4 (siANG#1 and #2), siNOX4, and SC siRNA (C, E) , or expression vectors of full-length (flANG), C-terminal (cANG), and N-terminal (nANG) ANGPTL4 (D), and then treated with or without 200 μM OA and 5 mM NAC for 16 h. Real-time quantitative PCR and western blotting were performed to examine NOX4 mRNA and ANGPTL4 protein levels, respectively (D). NS indicates not significant. The data are presented as the mean ± SEM. P -values were determined using a two-tailed Student's t -test. *** P < 0.001 (n=3).

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: Flow Cytometry, Staining, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: c-Jun expression is essential for OA- and ANGPTL4-induced NOX4 transcriptional activity. (A) Dual-luciferase reporter assay was performed to analyze the activation of NOX4 promoter in SW480 cells transfected with the RSV-c-Jun expression vector for 24 h. Firefly luciferase activity was determined and normalized to Renilla luciferase activity (i). Immunoblot analysis of cell lysates form SW480 cells was performed using anti-phospho-c-Jun Ser63 , anti-c-Jun, anti-NOX4, and anti-actin antibodies (ii). (B) Immunoblot analysis was performed using antibodies against HDAC1, c-Jun and phosphor-c-Jun Ser63 from SW480 cells transfected with 20 nM siANGPTL4 followed by treatment with 200 µM OA for 16 h (i). The secretion of ANGPTL4 was determined by ELISAs (ii). Chromatin immunoprecipitation (ChIP) assay (iii) and DNA affinity precipitation assay (iv) were performed to examine the binding of c-Jun to the NOX4 promoter with wild-type (WT) and mutated (mut) AP-1 sites in SW480 cells transfected with 20 nM siANGPTL4 followed by treatment with 200 µM OA for 16 h as described in “Materials and methods”. (C) Protein levels of ANGPTL4, NOX4, c-Jun Ser63 , c-Jun, and actin, NOX4 promoter activity, and the secretion of ANGPTL4 were determined by Immunoblot analysis (i), dual-luciferase reporter assay (ii) and ELISAs (iii) in cells transfected with 20 nM c-Jun siRNA (si-c-Jun) and expression vector of ANGPTL4 (flANG). The data are presented as the mean ± SEM. P -values determined using a two-tailed Student's t -test. *** P < 0.001 (n=3).

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: Expressing, Activity Assay, Luciferase, Reporter Assay, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Chromatin Immunoprecipitation, Affinity Precipitation, Binding Assay, Two Tailed Test

Journal: Theranostics

Article Title: Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

doi: 10.7150/thno.44744

Figure Lengend Snippet: The ANGPTL4/NOX4 axis is essential for OA-induced CRC extravasation and is associated with clinical outcome. (A) Tumor cells penetrate to pulmonary blood vessels that was determined by in vivo extravasation assay. DiI staining of SW480 cells were transfected with 20 nM siNOX4, siANGPTL4, siMMP-1, and siMMP-9 or treated with 5 mM NAC and then injected intravenously into the tail vein of 6-week-old SCID-NOD mice which were preinjected intravenously with OA at a final concentration of 200 µM (i). The gavage feeding of vitamin E (VitE; 100 mg/kg body weight) is scheduled as indicated (ii). At 48 h after injection of tumor cells, the mice were sacrificed for examining of metastatic tumor cells surrounding the lung tissue as described in 'Materials and Methods'. Tumor cell penetration was imaged using a microscope (iii). Original magnification, × 40 and × 100; DiI labeled tumor cells (red); CD31 labeled blood vessels (green); DAPI labeled nucleus (blue). The number of tumor cell extravasation was calculated by analyzing at least four sections and six fields (iv); Six mice were analyzed for each group. Real-time quantitative PCR analysis was performed to determine NOX4, ANGPTL4, MMP-1, and MMP-9 mRNA levels in SW480 cells treated with 200 µM OA for 48 h (v). Values are the mean ± SEM. *** P < 0.001, Student's t -test. (B) Box plots comparing the levels of ANGPTL4 and NOX4 mRNA in human CRC tissue (T) (n=65) and normal adjacent tissue (N) samples (n=65) were generated according to published data sets from Oncomine. *** P < 0.001, Student's t -test. Ref: Genes Chromosomes Cancer. 2010, 49:1024-34. (C) Kaplan-Meier curves showing CRC patient survival were retrieved from TCGA database. The median value was used to classify patients into high-expression or low-expression of ANGPTL4 (i) and NOX4 (ii). P values indicate the comparison between patients with high and low of ANGPTL4 or NOX4 expression. (D) Concurrent expression of ANGPTL4 and NOX4 in tumor tissues of CRC patients (n=597) in TCGA database was quantitated (Pearson's correlation coefficient is shown in the figures). FPKM: Fragments Per Kilobase of transcript per Million; Stages I~IV.

Article Snippet: Cell lines of colorectal cancer HCT116, SW480, SW620, LoVo, Colo205, DLD-1, and HT-29 were provided from Research Center of Clinical Medicine, National Cheng Kung University Hospital and Bioresource Collection and Research Center (BCRC, Hsinchu City, TW).

Techniques: In Vivo, Staining, Transfection, Injection, Concentration Assay, Microscopy, Labeling, Real-time Polymerase Chain Reaction, Generated, Expressing, Comparison

Journal: Journal of Gastrointestinal Oncology

Article Title: The roles and mechanisms of APOL1 in the development of colorectal cancer

doi: 10.21037/jgo-24-275

Figure Lengend Snippet: Effect of APOL1 on the biological behavior of CRC cells. (A) qRT-PCR was used to determine the APOL1 mRNA levels in HCT116, SW1116, and NCM460 cells. (B) In HCT116 cells, qRT-PCR was used to determine the interference efficacy of shAPOL1#1/2/3. (C) CCK8 assay was used to evaluate the proliferative capacity of HCT116 and SW1116 cells. (D) The count of clones in HCT116 and SW1116 cells was determined using the colony formation test. Staining method: crystal violet. Magnification: ×100. (E) Wound healing test was used to assess the migratory capacity of HCT116 and SW1116 cells (magnification: ×40). (F) The Transwell test was used to determine HCT116 and SW1116 cells’ invasion ability (magnification: ×100). Staining method: crystal violet. Each group, n=3. *, P<0.05 vs. NCM460 group; **, P<0.01; ***, P<0.001 vs . sh-NC group. sh-NC, shRNA-negative control; sh-APOL1, shRNA-apolipoprotein L1; OD, optical density; CRC, colorectal cancer; qRT-PCR, quantitative real-time polymerase chain reaction; CCK8, Cell Counting Kit 8.

Article Snippet: Shanghai iCell Bioscience Inc. (Shanghai, China) provided the human normal colon epithelial cells NCM460 (iCell-h373) and CRC cell lines [HCT116 (iCell-h071) and SW1116 cells (iCell-h201)].

Techniques: Quantitative RT-PCR, CCK-8 Assay, Clone Assay, Staining, shRNA, Negative Control, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Journal of Gastrointestinal Oncology

Article Title: The roles and mechanisms of APOL1 in the development of colorectal cancer

doi: 10.21037/jgo-24-275

Figure Lengend Snippet: Validation of the protein interaction of APOL1 with RUNX1. (A) In HCT116 and SW1116 cells, the protein interaction between APOL1 and RUNX1 was examined using a coimmunoprecipitation assay. (B) RUNX1 protein expression in HCT116 and SW1116 cells was assessed using western blotting. Each group, n=3. **, P<0.01; ***, P<0.001 vs . sh-NC group. IgG, immunoglobin G; sh-NC, shRNA-negative control; sh-APOL1, shRNA-apolipoprotein L1.

Article Snippet: Shanghai iCell Bioscience Inc. (Shanghai, China) provided the human normal colon epithelial cells NCM460 (iCell-h373) and CRC cell lines [HCT116 (iCell-h071) and SW1116 cells (iCell-h201)].

Techniques: Co-Immunoprecipitation Assay, Expressing, Western Blot, shRNA, Negative Control

Journal: Journal of Gastrointestinal Oncology

Article Title: The roles and mechanisms of APOL1 in the development of colorectal cancer

doi: 10.21037/jgo-24-275

Figure Lengend Snippet: RUNX1 overexpression affected EMT and the biological behavior of CRC cells by regulating the Wnt-β-catenin pathway. (A) Western blotting was used to detect the protein expression of RUNX1, β-catenin, cyclin D1, N-cadherin, vimentin, and E-cadherin. (B) The CCK-8 test was used to evaluate the proliferation of HCT116 and SW1116 cells, and the OD450 value was determined. (C) The wound healing test was used to assess the migratory potential of HCT116 and SW1116 cells (magnification: ×40). (D) The transwell test was used to characterize the invasion capacity in HCT116 and SW1116 cells (magnification: ×100). Staining method: crystal violet. Each group, n=3. **, P<0.01, ***, P<0.001 vs . sh-NC group; # , P<0.05; ## , P<0.01, ### , P<0.001 vs . sh-APOL1 + pcDNA-NC group. sh-NC, shRNA-negative control; sh-APOL1, shRNA- apolipoprotein L1; OD, optical density; EMT, epithelial-mesenchymal transition; CRC, colorectal cancer; CCK8, Cell Counting Kit 8.

Article Snippet: Shanghai iCell Bioscience Inc. (Shanghai, China) provided the human normal colon epithelial cells NCM460 (iCell-h373) and CRC cell lines [HCT116 (iCell-h071) and SW1116 cells (iCell-h201)].

Techniques: Over Expression, Western Blot, Expressing, CCK-8 Assay, Staining, shRNA, Negative Control, Cell Counting

Journal: Journal of Gastrointestinal Oncology

Article Title: The roles and mechanisms of APOL1 in the development of colorectal cancer

doi: 10.21037/jgo-24-275

Figure Lengend Snippet: Silencing the APOL1-mediated Wnt-β-catenin pathway affected EMT, proliferation, migration, and invasion in CRC cells. (A) Western blotting was used to measure the protein expression of RUNX1, β-catenin, cyclin D1, N-cadherin, vimentin, and E-cadherin. (B) CCK-8 assay was used to measure the proliferation of HCT116 and SW1116 cells, and the optical density at 450 nm value was determined. (C) Wound healing assay was used to assess the migratory potential of HCT116 and SW1116 cells (magnification: ×40). (D) The Transwell test was used to characterize the invasion capacity of HCT116 and SW1116 cells (magnification: ×100). Staining method: crystal violet. Each group, n=3. *, P<0.05, **, P<0.01, ***, P<0.001 vs . sh-NC group; # , P<0.05, ## , P<0.01, ### , P<0.001 vs . sh-APOL1 group. sh-NC, shRNA-negative control; sh-APOL1, shRNA-apolipoprotein L1; OD, optical density; EMT, epithelial-mesenchymal transition; CRC, colorectal cancer; CCK8, Cell Counting Kit 8.

Article Snippet: Shanghai iCell Bioscience Inc. (Shanghai, China) provided the human normal colon epithelial cells NCM460 (iCell-h373) and CRC cell lines [HCT116 (iCell-h071) and SW1116 cells (iCell-h201)].

Techniques: Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Staining, shRNA, Negative Control, Cell Counting