Figure 4: Effects of exogenous TRPV4 on human breast cancer cellular processes.

(a) Expression of wild-type human TRPV4 in stably retroviral-transduced breast cancer cell lines. Upper panel: Immunoblotting of exogenous TRPV4 and endogenous Actin (loading control) in whole-cell lysates. Lower panel: Immunoblotting of TRPV4 following Tunicamycin treatment (5 ug/mL), PNGaseF and Endo-H digestion (500 U/reaction). (b) Calcium imaging of TRPV4 stable transductants following treatment with 10 μM of 4α-PDD. (c) FACS analysis of MB468-TRPV4 stable transductants. Left panel: after gating on the live cells, single cells were gated using width and area parameters. Right panel: Histogram showing the percentages of cells in G1, S, and G2M phases. Average values (n = 3, ±s.d.) from two biological repeats and P values from unpaired Student’s t-test are shown. (d) Physical examination of TRPV4 stable transductants. Upper panel: Phase-contrast micrographs of the cell morphology of MB468 and MCF7 transduced with pBABE or pBABE-TRPV4 were obtained at sub-confluent density grown in standard medium containing 10% serum. Bottom left panel: relative cell surface area of control and TRPV4 stable transductants; bottom right panel: size distribution of control and TRVP4 stable transductants based on FSC-A data from FACS analysis and P values from unpaired Student’s t-test are shown (e) Effects of TRPV4 on the matrigel invasion and chemotaxis of MB468 cells transduced with pBABE or pBABE-TRPV4 retroviral particles using the Boyden chamber assays. Cells that migrated or invaded through the barrier were stained with hematoxylin/eosin and counted (for invasion assay) or subject to colormetric measurements (chemotaxis assay) in three independent experiments. Data are expressed as migrated/invaded cells per field (n = 3, means ± s.d.), P values from unpaired Student’s t-test are shown.