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: Heliyon

Article Title: A novel lnc-LAMC2-1:1 SNP promotes colon adenocarcinoma progression by targeting miR-216a-3p/HMGB3

doi: 10.1016/j.heliyon.2022.e12342

Figure Lengend Snippet: The lnc-LAMC2-1:1 SNP is a sponge of miR-216a-3p . Bar graphs of (a) and (b) show the relative luciferase activity of vectors containing SW480. A dual-luciferase reporter assay was performed, and the co-transfection of lnc-LAMC2-1:1 SNP and miR-216a-3p reduced the luciferase activity.

Article Snippet: The SW480 cell line was obtained from COSMOBIO Company in China and was grown in DMEM supplemented with 10% FBS (GIBCO) and 1% antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin) in a humidified atmosphere of 5% CO2 at 37 °C.

Techniques: Luciferase, Activity Assay, Reporter Assay, Cotransfection

Journal: Heliyon

Article Title: A novel lnc-LAMC2-1:1 SNP promotes colon adenocarcinoma progression by targeting miR-216a-3p/HMGB3

doi: 10.1016/j.heliyon.2022.e12342

Figure Lengend Snippet: The lnc-LAMC2-1:1 SNP positively regulates HMGB3 by sponging miR-126-3p (a) mRNA expression of HMGB3 in SW480 cells. Cells were transfected with mimics of NC, miR-126-3p mimics, ASO-NC, and ASO-miR-126-3p. (b) mRNA expression of HMGB3 in SW480 cells. Cells were transfected with pcDNA3.1, lnc-LAMC2-1:1 SNP , and lnc-LAMC2-1:1-wt. (c) Complementary sequences of HMGB3 and miR-126-3p in the StarBase database. (d) A dual-luciferase reporter assay was performed, and the co-transfection of miR-126-3p and HMGB3 reduced the luciferase activity.

Article Snippet: The SW480 cell line was obtained from COSMOBIO Company in China and was grown in DMEM supplemented with 10% FBS (GIBCO) and 1% antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin) in a humidified atmosphere of 5% CO2 at 37 °C.

Techniques: Expressing, Transfection, Luciferase, Reporter Assay, Cotransfection, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Radiosensitization to γ-Ray by Functional Inhibition of APOBEC3G

doi: 10.3390/ijms23095069

Figure Lengend Snippet: The enhancement ratios at 10% survival (ER 10 ) to cell lines.

Article Snippet: A375 (purchased from ATCC), DU145 (purchased from RIKEN BRC, Tsukuba, Japan), MDA-MB-231 (purchased from ATCC), MIAPaCa2 (obtained from National Cancer Center), SAS (purchased from JCRB Cell Bank, Osaka, Japan), SW480 (purchased from JCRB Cell Bank), and U2OS (purchased from ATCC) cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco).

Techniques: