Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: For immunofluorescence staining, antibodies against the following proteins were used: phosphorylated FAK(Y397) (1:300, 44-624G), CHC (1:500, X22, MA1-065), paxillin (1:300, 612405) from BD Pharmingen, CHC (1:500, ab21679), paxillin (1:300, ab32084) from Abcam, phosphorylated FAK(Y397) (1:200, MAB4528) from RD Systems, WAVE2 (1:200, sc-373889 or 1:200, #3659) from Santa Cruz Biotechnology and Cell Signaling, respectively, and cortactin (1:200, #3503) from Cell Signaling.

Techniques: Transfection, Western Blot

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa knockdown inhibits recruitment of FAK and paxillin to, and maturation of, FAs. (A) siRNA-transfected HeLa cells were plated on collagen IV-treated glass coverslips for 15–120 min, and fixed and stained with anti-pFAK(Y397) (pFAK, green) and -paxillin (red) antibodies. Single confocal sections near the adherent surface were obtained as described in the Materials and Methods. Scale bar: 10 µm. (B) Representative x-y image planes near the adherent surface, with y-z projections on the right, are shown for cells processed as in A. Scale bars: 5 µm. (C) Quantification of the number of mature (longer than 2 µm) FAs per cell in siRNA-transfected HeLa cells plated on collagen IV coverslips for 15–120 min. The graph represents a summary of three experiments. **P<0.005. (D) Representative TIRF microscopy images of phosphorylated FAK (pY397) in siRNA-transfected cells processed as in A. Scale bar: 5 µm.

Article Snippet: For immunofluorescence staining, antibodies against the following proteins were used: phosphorylated FAK(Y397) (1:300, 44-624G), CHC (1:500, X22, MA1-065), paxillin (1:300, 612405) from BD Pharmingen, CHC (1:500, ab21679), paxillin (1:300, ab32084) from Abcam, phosphorylated FAK(Y397) (1:200, MAB4528) from RD Systems, WAVE2 (1:200, sc-373889 or 1:200, #3659) from Santa Cruz Biotechnology and Cell Signaling, respectively, and cortactin (1:200, #3503) from Cell Signaling.

Techniques: Transfection, Staining, Microscopy

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa knockdown decreases the colocalization of clathrin and pFAK in the lamellipodial region of HeLa cells. siRNA-transfected cells were plated on collagen IV for 1 h, fixed and co-stained anti-pFAK(Y397) (phFAK) and CHC (ab21679) antibodies. Representative deconvolved confocal images are shown, with arrowheads in NC indicating areas containing pFAK staining in close proximity to fine clathrin puncta. Enlargements of the boxed areas in NC and KD-a cells are shown underneath. The experiment was performed three times. Scale bars: 10 µm; 2 µm in insets.

Article Snippet: For immunofluorescence staining, antibodies against the following proteins were used: phosphorylated FAK(Y397) (1:300, 44-624G), CHC (1:500, X22, MA1-065), paxillin (1:300, 612405) from BD Pharmingen, CHC (1:500, ab21679), paxillin (1:300, ab32084) from Abcam, phosphorylated FAK(Y397) (1:200, MAB4528) from RD Systems, WAVE2 (1:200, sc-373889 or 1:200, #3659) from Santa Cruz Biotechnology and Cell Signaling, respectively, and cortactin (1:200, #3503) from Cell Signaling.

Techniques: Transfection, Staining

Journal: Cells

Article Title: Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

doi: 10.3390/cells10061484

Figure Lengend Snippet: Effects of XN on the activity/expression of MMP-9 and MMP-2 in PMA-stimulated A549 cells. The cells were pre-incubated with XN for 4 h, followed by PMA co-stimulation for 24 h. ( a ) The gelatinolytic activity of MMP-9 and MMP-2 was assayed using gelatine zymography. The concentrations of MMP-9 ( b ) and MMP-2 ( c ) in the supernatants were determined with the ELISA assay. The results are the mean ± SD of three independent experiments, * p < 0.05, *** p < 0.001 in comparison to the PMA-only treatment; ### p < 0.001 in comparison to the non-stimulated cells; one-way ANOVA test. ( d ) The levels of MMP-9 and MMP-2 protein expression from whole cell lysates were measured with the Western blot analysis. β-actin was used as an internal control. The intensity of bands for MMP-9 ( e ) and MMP-2 ( f ) from the Western blot was quantified by densitometry analysis, where the untreated PMA-induced cells represented 100%. The results are the mean ± SD of three independent experiments, * p < 0.05, *** p < 0.001 in comparison to the PMA-only treatment, ### p < 0.001 in comparison to the non-stimulated cells; one-way ANOVA test.

Article Snippet: Phospho-FAK was assessed with a Human phospho-FAK (Y397) ELISA Kit (Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China).

Techniques: Activity Assay, Expressing, Incubation, Zymography, Enzyme-linked Immunosorbent Assay, Comparison, Western Blot, Control

Journal: Cells

Article Title: Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

doi: 10.3390/cells10061484

Figure Lengend Snippet: Effect of XN on the endogenous and exogenous production of TIMP-1 and TIMP-2 in PMA-induced A549 cells. ( a ) After the 4-h pre-treatment with XN followed by 24-h co-incubation with PMA, the levels of TIMP-1 and TIMP-2 were determined by immunoblotting. ( b ) The intensity of protein bands from Western blot was quantified by densitometry analysis, where the untreated PMA-induced cells represented 100%. The results are presented as the mean ± SD of three independent experiments, ** p < 0.01, in comparison to the PMA-only treatment, ## p < 0.01 in comparison to the non-stimulated cells; one-way ANOVA test. ( c ) Following the XN treatment, the supernatants from the A549 cell cultures were collected and the levels of TIMP-1 and TIMP-2 were assayed with ELISA. The results are the mean ± SD of three independent experiments, *** p < 0.001 in comparison to the PMA-only treatment, ### p < 0.001 in comparison to the non-stimulated cells; one-way ANOVA test.

Article Snippet: Phospho-FAK was assessed with a Human phospho-FAK (Y397) ELISA Kit (Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China).

Techniques: Incubation, Western Blot, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

doi: 10.3390/cells10061484

Figure Lengend Snippet: Effect of XN on VEGF and TGF-β release from PMA-stimulated cells. The A549 cells were pre-incubated with the indicated concentrations of XN for 4 h and then co-incubated in the presence of PMA for 24 h. Following the treatment, the concentration of VEGF ( a ) and TGF-β ( b ) in the supernatants was assessed using the ELISA assay. The results are presented as the mean ± SD of three independent experiments, ** p < 0.01, *** p < 0.001 in comparison to the PMA-only treatment, ### p < 0.001 in comparison to the non-stimulated cells; one-way ANOVA test.

Article Snippet: Phospho-FAK was assessed with a Human phospho-FAK (Y397) ELISA Kit (Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cells

Article Title: Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

doi: 10.3390/cells10061484

Figure Lengend Snippet: XN inhibits the progress of epithelial-mesenchymal transition in PMA-induced A549 cells. ( a ) The A549 cells were pre-treated with XN at the indicated concentrations for 4 h prior to 24-h co-stimulation with PMA (50nM). Next, they were subjected to Western blotting to analyse the levels of vimentin, α-catenin, E-cadherin, and N-cadherin proteins. β-actin was used as an internal control. ( b ) The intensity of protein bands from immunobloting was quantified by densitometry analysis, where the untreated PMA-induced cells represented 100%. The results are the mean ± SD of three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the PMA-only treatment, ### p < 0.001 in comparison to the non-stimulated cells; one-way ANOVA test. ( c ) After the treatment with the selected concentrations of XN followed by PMA stimulation, the cells were incubated with anti-vimentin antibody or anti-α-catenin antibody followed by FITC labelled secondary antibody. The subcellular identification of vimentin and α-catenin was performed under a fluorescence microscope at 200 x magnification. ( d ) XN decreases the expression of the Snail-1 transcription factor. After the treatment, the amounts of the Snail-1 protein in the total cell extracts were assessed with the ELISA assay. The results are the mean ± SD of three independent experiments. *** p < 0.001 compared to the PMA-only treatment, ### p < 0.001 in comparison to the non-stimulated cells; one-way ANOVA.

Article Snippet: Phospho-FAK was assessed with a Human phospho-FAK (Y397) ELISA Kit (Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China).

Techniques: Western Blot, Control, Comparison, Incubation, Fluorescence, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

doi: 10.3390/cells10061484

Figure Lengend Snippet: XN interferes with PMA-dependent activation of AKT, FAK, and ERK1/2, kinases in A549 cells. The cells were pre-treated with XN for 4 h and then co-treated with PMA for 24 h; the amounts of phosphorylated ERK1/2, FAK, and AKT were determined with the ELISA assay. Quantitative analysis of ( a ) p-AKT/tAKT, ( b ) p-FAK/tFAK, and ( c ) p-ERK/tERK ratio. Data are expressed as means ± SD of three independent experiments. *** p < 0.001 compared to the PMA-only treatment, # p < 0.05 or ### p < 0.001 compared to the non-stimulated cells; one-way ANOVA test.

Article Snippet: Phospho-FAK was assessed with a Human phospho-FAK (Y397) ELISA Kit (Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

doi: 10.3390/cells10061484

Figure Lengend Snippet: The XN-mediated suppression of the PMA-induced invasive potential of A549 cells and MMP-9 production is associated with alteration of FAK/AKT and MAPK/ERK signalling pathways. The A549 cells were exposed to a single treatment (5 µM XN, LY294002, Y15, or SC772984) or a combination treatment (XN + Ly294002, XN + Y15, or XN + SC772984) for 4 h, and then incubated in the presence of PMA (50 nM) for 24 h. ( a ) The invasive ability of the treated cells was studied using the Transwell in vitro invasion assay. The invasion of the untreated PMA-stimulated cells was established as 100%. *** p < 0.001 compared to the PMA-stimulated cells; ### p < 0.001; one-way ANOVA test. ( b ) After the single or combination treatment (as indicated above), the supernatants from the PMA-stimulated cells were subjected to the ELISA assay to analyse the levels of the MMP-9 protein. The results are the mean ±SD from three independent experiments. *** p < 0.001 compared to the PMA-treated cells, ### p < 0.001; one-way ANOVA test.

Article Snippet: Phospho-FAK was assessed with a Human phospho-FAK (Y397) ELISA Kit (Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China).

Techniques: Incubation, In Vitro, Invasion Assay, Enzyme-linked Immunosorbent Assay