To determine if elevated expression of the MT1 MLT receptor could enhance the sensitivity of MCF-7 cells to MLT and enhance MLT mediated expression of ARH1, MCF-7 cells were treated with diluent (0.01% ethanolic cell culture media), MLT (10 nM), trichostatin A (TSA, 0.7 μM), or TSA + MLT for 24 h. Cells were then harvested in lysis buffer after and total cellular protein isolated and analyzed by Western blot analysis for the expression of MT1 (ab87639, Abcam, Cambridge, MA) and ARHI protein levels as described above.
Twenty four hours after treatment with TSA (TSA, 0.7 μM) or diluent (0.01% ethanolic media) paclitaxel-resistant MCF-7 cells (MCF-7/PAC, gift from Dr. Timothy Grant, University of Leicester, UK) were plated at a density of 20 × 105 cells per ml in six-well plates in IDMEM supplemented with 10% FBS45 (link). Five hours after seeding, cells were treated with PTX (uM ),PTX, TCA (0.7 μM), MLT (10 nM), or TCA + MLT. On specific days (1, 3, 5, or 7), cells were trypsinized, mixed with 2% trypan blue, and total and viable cells counted on a haemocytometer.
Twenty four hours after treatment with TSA (TSA, 0.7 μM) or diluent (0.01% ethanolic media) paclitaxel-resistant MCF-7 cells (MCF-7/PAC, gift from Dr. Timothy Grant, University of Leicester, UK) were plated at a density of 20 × 105 cells per ml in six-well plates in IDMEM supplemented with 10% FBS45 (link). Five hours after seeding, cells were treated with PTX (uM ),PTX, TCA (0.7 μM), MLT (10 nM), or TCA + MLT. On specific days (1, 3, 5, or 7), cells were trypsinized, mixed with 2% trypan blue, and total and viable cells counted on a haemocytometer.