epidermoid human cancer cell line a431  (ATCC)

Journal: Cellular and molecular life sciences : CMLS

Article Title: Tetraspanin-enriched Microdomains Regulate Digitation Junctions

doi: 10.1007/s00018-018-2803-2

Figure Lengend Snippet: A. Role of PI-4 kinase signaling in DJs. PI4P and EWI2/PGRL are colocalized in DJs and memtubs of A431 cells. B. Role of PI-3 kinase signaling in DJs. Du145 cells cultured on glass coverslips were treated with wortmannin (100 nM) in serum-free medium at 37oC for 60 mins before they were fixed, incubated with CD9 mAb, DAPI, and phalloidin, and examined with confocal microscopy. C. Role of MLCK signaling. Du145 cells cultured on glass coverslips were treated with ML-7 (30 μM) in serum-free medium at 37°C for 60 mins before they were fixed, incubated with CD9 or CD44 mAbs, DAPI, and phalloidin, and then examined with confocal microscopy. Quantification of microextrusion density in DJs (mean±S.E, n=3 individual experiments). ***: P<0.001. Scale bars: 10 μm.

Article Snippet: Cells used in this study include human microvascular endothelial cells (HMECs) (CDC, Atlanta, GA), Du145 and LnCap human prostate cancer (ATCC, Manassas, VA), U87 human glioblastoma (ATCC), and A431 human epidermoid cancer cell lines (ATCC), Du145-Mock and -CD82 stable transfectants 18 , 48 , HT1080-Mock and HT1080-CD9 stable transfectants, PC3 human prostate cancer cells (ATCC) in which EWI2/PGRL was transiently silenced with siRNA oligo against the target sequence GUUCUCCUAUGCUGUCUU of EWI2/PGRL or in which control siRNA oligo was transfected 22 , MDCK-GFP:CD82 and -GFP:CD151 stable transfectants expressing the fusion proteins in which eGFP moieties were fused to the N-termini of CD82 and CD151 proteins, respectively, and NIH3T3-EWI2/PGRL transfectant 25 .

Techniques: Cell Culture, Incubation, Confocal Microscopy

Journal: Scientific Reports

Article Title: CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion

doi: 10.1038/s41598-018-35792-8

Figure Lengend Snippet: CASP4 -silencing in epithelial cancer cell lines inhibits cell migration. ( a ) Western blot analysis of CASP4 and tubulin expression in A431 cells transfected with the indicated siRNA (100–200 nM). siCTRL was luciferase siRNA in all experiments. ( b ) Analysis of A431 cell migration by wound healing assays. The wound closure was quantified at 8 hours post-wound by measuring the cell-free area using the ImageJ software . Bar plots represent the percentage of relative wound closure (on the left) and migration velocity (on the right), calculated as described in materials and methods. Data were obtained by the analysis of 11 microscopy images and they are representative of two independent experiments (relative wound closure: p < 0.0001, n = 11; migration velocity: p < 0.0001, n = 44 corresponding to 4 length values/image). ( c ) Analysis of A431 transwell cell migration assays. Bar plots represent the optical reading (OD 570 nm ) of FBS-guided cell migration of siCTRL and siCASP4 transfected cell lines (p = 0.01, n = 5). The data are representative of two independent experiments. ( d ) Western blot analysis of CASP4 and tubulin expression in NSCLC cells transfected with siCTRL and siCASP4 #1 at 100 nM concentration. ( e ) Analysis of NSCLC transwell cell migration assays. Bar plots represent the optical reading (OD 570 nm ) values of FBS-guided cell migration of siCTRL and siCASP4 #1 transfected cell lines allowed to migrate for 15–20 hours (A549: p = 0.008, n = 5; HCC827: p = 0.0004, n = 9; HCC4006: p = 0.003, n = 11; H1650 and H1975: p < 0.0001, n = 11 and n = 14). The data are representative of three independent experiments. Statistical analysis was performed by Wilcoxon rank sum test for the comparisons of siCASP4 with the siCTRL transfected cell lines. Significant p-values are represented by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Non-significant p-values are not shown.

Article Snippet: Human epidermoid A431 cancer cell line, lung carcinoma A549 cell line (ATCC), Phoenix TM Retroviral Packaging Cell Lines Ampho (ATCC, Manassas, VA, USA) and human kidney 293FT cell line (Thermo Fisher Scientific, Waltham, MA USA) were cultured in Dulbecco’s Modified Eagle’s Medium, 4.5 g/L glucose (DMEM) (BioWhittaker, Lonza, Italy); non small cell lung cancer (NSCLC) HCC827, HCC4006, H1650 and H1975 cell lines, kindly provided by Dr Oreste Segatto, Regina Elena National Cancer Institute, Rome (Italy), and RC2.2 an erlotinib-resistant NSCLC mesenchymal cell line derived from the erlotinib-sensitive HCC4006 were cultured in RPMI 1640 (BioWhittaker, Lonza, USA).

Techniques: Migration, Western Blot, Expressing, Transfection, Luciferase, Software, Microscopy, Concentration Assay

Journal: Scientific Reports

Article Title: CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion

doi: 10.1038/s41598-018-35792-8

Figure Lengend Snippet: Effects of CASP4 -silencing on cell-cell junctions and actin cytoskeleton polymerization. Representative confocal microscopy images of A431 cells transfected with siCTRL (A–C) or siCASP4 (D–F) stained with E-cadherin reactive antibody (red) (A,D) and phalloidin (green) (B,E) at the leading edge ( a ) and in the underneath confluent cell monolayer ( b ); merged pictures (C,F) are shown. Optical 2x zoom (A’–F’) were analyzed with the yz plane projections. Bar plots indicate the percentage of fully sealed junctions at leading edge (p = 0.0003, n = 10). E-cadherin positive junctions were analyzed in 10 confocal microscopy images recorded in two independent experiments; approximately 500 junctions were counted by using ImageJ. In panel (b) both xz and yz planes are shown. Scale bars (25 µm) are indicated. Statistical analysis was performed by Wilcoxon rank sum test for the comparison of siCASP4 with the siCTRL transfected A431 cells. Significant p-values are represented by asterisks: ***p < 0.001.

Article Snippet: Human epidermoid A431 cancer cell line, lung carcinoma A549 cell line (ATCC), Phoenix TM Retroviral Packaging Cell Lines Ampho (ATCC, Manassas, VA, USA) and human kidney 293FT cell line (Thermo Fisher Scientific, Waltham, MA USA) were cultured in Dulbecco’s Modified Eagle’s Medium, 4.5 g/L glucose (DMEM) (BioWhittaker, Lonza, Italy); non small cell lung cancer (NSCLC) HCC827, HCC4006, H1650 and H1975 cell lines, kindly provided by Dr Oreste Segatto, Regina Elena National Cancer Institute, Rome (Italy), and RC2.2 an erlotinib-resistant NSCLC mesenchymal cell line derived from the erlotinib-sensitive HCC4006 were cultured in RPMI 1640 (BioWhittaker, Lonza, USA).

Techniques: Confocal Microscopy, Transfection, Staining, Comparison

Journal: Scientific Reports

Article Title: CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion

doi: 10.1038/s41598-018-35792-8

Figure Lengend Snippet: Characterization of stable silenced A431-derived LR cell lines. ( a ) Western blot analysis of CASP4 and tubulin expression in LR cell lines. ( b , c ) Cell migration analysis: ( b ) wound closure was quantified in 4–6 images for the indicated cell lines at 8–12 h post-wound. Bar plots represent the percentage of relative wound closure (LR1.2 - LR3.2: p = 0.002, n = 6; LR1.2 - LR4.2: p = 0.009, n = 4); ( c ) transwell cell migration analysis. Bar plots represent the optical reading (OD 570 nm ) of FBS-guided cell migration (LR1.2 - LR3.2: p = 0.002, n = 6; LR1.2 - LR4.2: p = 0.005, n = 6). Data are representative of three independent experiments. ( d ) Analysis of cell migration by wound healing assays in retroviral infected-cells. The wound closure was quantified in 12 images for the indicated cell lines at 6 hours post-wound. Bar plots indicate the percentage of wound closure in the infected LR1.2 cells (FLAG-CASP4.C258S and FLAG-CASP4, p < 0.0001, n = 12). Data are representative of three independent experiments. ( e ) Bar plots indicate the percentage of FLAG + cells in FLAG-CASP4 infected LR1.2 cell line counted in 13 different fields in two independent experiments by using ImageJ (p < 0.0001, n = 13). Statistical analysis was performed by Wilcoxon rank sum test for the comparison of LR3.2 and LR4.2 with LR1.2 cell lines, and with t-test for the comparison of FLAG-CASP4 with control cells, and for the comparisons of percentage of the FLAG + cells. Significant p-values are represented by asterisks: **p < 0.01, ****p < 0.0001. Non-significant p-values are not shown.

Article Snippet: Human epidermoid A431 cancer cell line, lung carcinoma A549 cell line (ATCC), Phoenix TM Retroviral Packaging Cell Lines Ampho (ATCC, Manassas, VA, USA) and human kidney 293FT cell line (Thermo Fisher Scientific, Waltham, MA USA) were cultured in Dulbecco’s Modified Eagle’s Medium, 4.5 g/L glucose (DMEM) (BioWhittaker, Lonza, Italy); non small cell lung cancer (NSCLC) HCC827, HCC4006, H1650 and H1975 cell lines, kindly provided by Dr Oreste Segatto, Regina Elena National Cancer Institute, Rome (Italy), and RC2.2 an erlotinib-resistant NSCLC mesenchymal cell line derived from the erlotinib-sensitive HCC4006 were cultured in RPMI 1640 (BioWhittaker, Lonza, USA).

Techniques: Derivative Assay, Western Blot, Expressing, Migration, Retroviral, Infection, Comparison, Control

Journal: Scientific Reports

Article Title: CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion

doi: 10.1038/s41598-018-35792-8

Figure Lengend Snippet: CASP4 -silencing impairs cell detachment and influences number and size of focal adhesions. ( a,b ) Percentage of cells detached from tissue culture plate upon treatment with trypsin/EDTA is shown in y-axis. The percentage is calculated with respect to untreated LR1.2, LR3.2 and LR4.2 cell lines ( a ) and untreated siCTRL and siCASP4 transfected A431 cells ( b ). Time (min) of incubation in detachment solution is reported in x-axis. Data are representative of three independent experiments (LR1.2 - LR3.2 and LR1.2 - LR4.2 at 2, 4, 10 and 15 min, p = 0.05, n = 3–6; siCASP4 - siCTRL: 4 min, p = 0.04; 10 min, p = 0.05, n = 3). ( c ) Representative confocal microscopy images of talin-stained (red) cells, cultured on poly-L-lysine treated glasses; 4x enlargments are also shown, corresponding to the yellow boxes in LR1.2 (A), LR3.2 (B) and LR4.2 (C). The bar plots indicate the percentage of focal adhesions (FA) positive cells (n = 34–38), the number of FA per cell (n = 39–42) and the FA length (n = 236–597) calculated in clusters of 6–10 cells present in 7–10 fields from three independent experiments (FA positive cells, FA number/cells, FA length: LR1.2 - LR3.2 and LR1.2 - LR4.2, p < 0.0001; FA number/cells: LR3.2 - LR4.2, p = 0.03). Scale bars are indicated. Statistical analysis for every pair-comparison was performed by Wilcoxon rank sum test. Significant p-values are represented by asterisks: *p < 0.05; ****p < 0.0001. Non-significant p-values are not shown.

Article Snippet: Human epidermoid A431 cancer cell line, lung carcinoma A549 cell line (ATCC), Phoenix TM Retroviral Packaging Cell Lines Ampho (ATCC, Manassas, VA, USA) and human kidney 293FT cell line (Thermo Fisher Scientific, Waltham, MA USA) were cultured in Dulbecco’s Modified Eagle’s Medium, 4.5 g/L glucose (DMEM) (BioWhittaker, Lonza, Italy); non small cell lung cancer (NSCLC) HCC827, HCC4006, H1650 and H1975 cell lines, kindly provided by Dr Oreste Segatto, Regina Elena National Cancer Institute, Rome (Italy), and RC2.2 an erlotinib-resistant NSCLC mesenchymal cell line derived from the erlotinib-sensitive HCC4006 were cultured in RPMI 1640 (BioWhittaker, Lonza, USA).

Techniques: Transfection, Incubation, Confocal Microscopy, Staining, Cell Culture, Comparison