br Analysis of protein expression by western blot br Followi
2.8. Analysis of protein expression by western blot
Following cell lysis, proteins were resolved using SDS–PAGE, transferred overnight to PVDF membranes (Bio-Rad Laboratories, USA), then incubated with primary and secondary antibodies. Primary anti-bodies for VEGF, NF-κB p65, NF-κB p50, IκB-α, p-IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA), Cx43 (Invitrogen, USA), IL-1β, TNF-α, and GAPDH (Abcam, USA) were used. Protein bands were visualized by chemiluminescence and quantified by densitometry using Image Lab Software (Bio-Rad Laboratories, USA).
2.9. Enzyme-linked immunosorbent assay
The levels of IL-1β, TNF-α, and VEGF secreted from cells treated or not with Av were measured using specific ELISA kits (BD Biosciences, USA).
MDA-MB-231 and MDA-Cx43D treated or not with Av were fixed, permeabilized and incubated with NF-κB p65, IκB-α or p-IκB-α primary Galactose 1-phosphate followed by secondary antibodies. Nuclei were counter-stained with Hoechst 33342 (Eugene, USA) and samples were imaged using a laser scanning confocal microscope (LSM 710, Carl Zeiss, Germany).
2.11. Immunofluorescence staining of paraﬃn-embedded tissues
Lung tissue sections were obtained from control or Av-treated mice. Tissues, 5-μm thick, were immunostained for IκB-α and p-IκB-α ex-pression. Sections were heated to 50 °C for 40 min, deparaﬃnized in xylol, and then rehydrated. Following antigen retrieval sections were washed with deionized water and blocked with 5% normal goat serum in PBS for 1 h in a humidified chamber. Sections were incubated with primary antibodies overnight at 4 °C, followed by washing and in-cubation with IgG-conjugated secondary antibody (Alexa 488 and Texas Red). The sections were then washed with PBS, mounted with Prolong Anti-fade, and observed under the fluorescent microscope (LSM 710, Carl Zeiss, Germany).
2.12. Gelatin zymography
Proteins extracted from control or Av-treated cells were resolved on SDS-PAGE gel containing gelatin as a substrate. Gels were incubated in substrate buﬀer overnight at 37 °C. Gels were then stained with Coomassie blue (R250) and visualized using Chemidoc MP Imaging System. Band intensity was analyzed using Image Lab Software (Bio-Rad Laboratories, USA).
2.13. Real-time migration, invasion and proliferation assay
Quantitative analysis of the eﬀect of Av treatment on the migration, invasion and proliferation of MDA-MB-231 and MDA-Cx43D was per-formed with slight modifications using xCELLigence Real-time Cell Analyzer (RTCA, Roche Applied Science, USA) . Control or Av-treated cells were re-suspended in serum-free media and seeded in the upper chamber of the RTCA CIM-plates, coated with Matrigel for in-vasion assays or left uncoated for the migration assays. For proliferation assays, cells were seeded in E-plates. Migration, invasion and pro-liferation were monitored every 15 mins for a minimum of 18 h, by recording cell impedance produced as the cells attached and detached from the gold electrodes in the CIM and E-plates. The generated cell index (CI) correlates directly with cell number. Data were expressed as bar graphs of CI % of control.
2.14. Statistical analysis
Results are expressed as individual data or as the average ± SEM. Statistical comparisons were performed using Student's t-test or ANOVA in order to determine statistical significance. P value was determined and significance level was set at P < .05. Microsoft Excel and GraphPad Prism 7 were used to perform statistical analysis.
Fig. 4. Av treatment increases mRNA levels of EMT markers and decreases mRNA levels of MET marker, E-Cadherin. (A) qPCR showing the eﬀect of Av treatment of MDA-MB-231 cells on Twist mRNA ex-pression (B) Snail (C) N-Cadherin and (D) E-Cadherin. Data foreach target mRNA were normal-ized to GAPDH. Results are representative of three diﬀerent independent experiments. *, **, *** in-dicate P < .05, P < .001, P < .0001, respectively.
Fig. 5. Av treatment increases mRNA levels and ac-tivity of MMP2 and MMP9. qPCR showing the eﬀect of Av treatment in MDA-MB-231 cells on transcrip-tional levels of MMP2 (A) and MMP9 (B). Data for each target mRNA were normalized to GAPDH. (C) Gelatin zymography of Av-treated MDA-MB-231 cells. FBS was used as an internal control. Proteins were separated on a gel containing gelatin, the sub-strate of MMPs, in order to assess the activation status and levels of these enzymes. Densitometry quantification of MMP2 and MMP9 bands was then performed using Image Lab software. Quantification of each band was normalized to control. (D) Invasion of MDA-MB-231 cells. Quantification graphs nor-malized cell index values, relative to controls, for 24 h pre-treatment. Cell impedance readings were taken every 15 min for a minimum of 18 h. Results are representatives of two independent experiments (n = 2). Results are representative of three diﬀerent independent experiments. *, **, *** indicate P < .05, P < .001, P < .0001, respectively.