Figure 5

A. Total number of puromycin-resistant colonies generated after supertransfection of episomal vectors expressing Rhox2, Rhox4, anti-sense Rhox2, anti-sense Rhox4 or a control empty vector. Mann Whitney test revealed significant differences between control and Rhox2 sense (P < 0.03) and control and Rhox4 sense (P < 0.03). B. Alkaline phosphatase stained ES cell cultures after supertransfection with either an empty episomal vector (vector) or an Egfp-, Rhox4-, antisense Rhox4, Rhox2- or antisense Rhox2- expressing episomal vector. Transfected cells were selected in puromycin for 6 days and subsequently replating in the presence (+) and absence (-) of LIF. C. Western blot analysis of COS7 cells transiently co-transfected with vectors expressing sense (S) Rhox2 (lane 3 and 5) or Rhox4 (lanes 2, 4 and 6) and vectors expressing anti-sense (AS) Rhox2 (lanes 1 and 2) or anti-sense Rhox4 (lanes 3 and 4) showing efficient knockdown of RHOX4 protein product in the presence of anti-sense Rhox4 (lane 4) but not anti-sense Rhox2 (lane2). Blots were stripped and re-probed with GAPDH to ensure equivalent loading.