Decrease in the HS4C3-binding epitope inhibits differentiation and apoptosis.

<p> (A) and (J) Real time PCR analysis of <i>3OST-5</i> in stable (A) and transient (J) <i>3OST-5</i> KD cells. The values shown are means ± SD after normalization against control cells (arbitrary value = 1). (B) FACS analysis using the anti-HS antibody HS4C3 (<i>black line</i>, control ESCs; <i>blue line,</i> stable <i>3OST-5-1</i> KD ESCs; <i>red line</i>, control EBs; <i>green line</i>, stable <i>3OST-5-1</i> KD EBs). The control cells were transfected with the construct that targeted <i>EGFP</i>. The <i>gray line</i> shows the result obtained for cells not treated with primary antibody. (C) Self-renewal assay with stable <i>3OST-5</i> KD cells. <i>Left panels</i> show photographs of representative colonies. Scale bars, 200 µm. The <i>right panel</i> shows the proportion of AP-positive colonies. The values shown are the mean ± SD. (D) and (E) Real time PCR analysis of marker genes in stable <i>3OST-5</i> KD cells at 6 days after LIF withdrawal (D) and at 3 (<i>Fgf5</i>, <i>Goosecoid</i>, and <i>Sox17</i>), 4 (<i>Nanog</i> and <i>Oct3/4</i>), or 12 (<i>Pax6</i>) days after EB formation (E). The values shown are means ± SD after normalization against control cells (arbitrary value = 1). (F) Western blot analysis, using anti-pErk1/2 and Erk1/2 antibodies, of cells stimulated with Fgf4. The histograms show mean densitometric readings ± SD expressed as the ratio p-Erk1/2/Erk1/2. Representative results are shown. (G) Real time PCR analysis of marker genes, <i>Fgf5</i> and <i>Goosecoid</i>, in cells treated with IETD at 3 days after EB formation. The values shown are means ± SD after normalization against nontreated cells (arbitrary value = 1). (H) FACS analysis using the anti-HS antibody HS4C3 in cells overexpressing <i>3OST-5</i> at 2 days after EB formation. The values shown are mean fluorescence intensity ± SD. (I) Real time PCR analysis of <i>Fgf5</i> and <i>Goosecoid</i> in cells overexpressing <i>3OST-5</i> at 0–4 days after EB formation (<i>black line</i>, control cells; <i>red line</i>, cells overexpressing <i>3OST-5</i>). The values shown are means ± SD from duplicate measurements from one representative experiment. (K) FACS analysis using the anti-HS antibody HS4C3 (<i>black line</i>, control cells; <i>red line</i>, transient <i>3OST-5-2</i> KD cells). The <i>gray line</i> shows the result obtained for cells not treated with primary antibody. (L) Measurement of apoptosis in transient <i>3OST-5</i> KD cells using an annexin V-FITC kit at 2 days after transfection. The values shown are the means ± SD after normalization against control cells in the absence of LIF (arbitrary value = 1). KD, knockdown; RNAi, RNA interference; EB, embryoid body. *, <i>P</i><0.01; **, <i>P</i><0.05. Three independent experiments were performed in each case.