Figure 3
From: MITF and BRN2 contribute to metastatic growth after dissemination of melanoma
Impact of MITF or BRN2 knockdown on cellular proliferation in vitro or in vivo. (a) MM649 or (b) HT144 cells were seeded into 96-well plates, induced with doxycycline for 7 days and a Sulforhodamine B assay was used to measure cellular proliferation rate. Values indicate mean +/− SD. *P < 0.05, unpaired t-test. (c) Changes in the cell cycle rate were measured by incubation of Cell Trace Violet stained cells for 6 days in the presence of doxycycline and measuring the change in staining by FACS. SED% positive was used to quantitatively measure changes in cell staining. **P < 0.01, unpaired t-test. (d) MM649 or (e) HT144 cells with inducible depletion of MITF or BRN2 were injected sub-cutaneously into the right and left flanks of 5-week-old male BALB/c Foxn1 nu (nude) mice, one shRNA per mouse. Expression of shRNA in established tumors was induced by the addition of doxycycline to drinking water when tumors reached approximately 50 mm3 (nominated Day 0). Values indicate mean +/− SEM, n = 5 mice per group, 10 tumors in total for MM649; n = 6 mice per group, 12 tumors in total for HT144. *P < 0.05, ****P < 0.0001, Mann-Whitney test. (f) Western blot analysis of MITF and BRN2 expression in MM649 tumors on completion of the experiment (Day 20 post-doxycycline treatment). Black shapes, vehicle only; white shapes, doxycycline. SD, standard deviation; SEM, standard error of the mean. Full-length blots are presented in Supplementary Figure S11.
