Fig. 7

Genetic interference of Cdkn1a, a canonical target of Sall2. a Left: Representative Western blot for SALL2, P21, and ACTIN in Sall2 WT and KO cells. Right: Quantification of P21 protein bands normalized to ACTIN (N = 3, *** P < 0.001 versus WT; Student’s T-test.). b Cdkn1a mRNA levels in Sall2 WT versus KO iMEFs. We isolated, reverse transcribed and analyzed RNA from WT or Sall2-null iMEFs by quantitative real-time PCR. Shown are the expression levels normalized to the Polr2A gene for Cdkn1a when compared to levels in Sall2 WT iMEFs. We expressed the values as fold change from WT iMEFs (N = 3, * P < 0.05 versus WT; Student’s T-test). c Left: Same analysis as in (a) from scramble (shCtrl) and Sall2-silenced cells (shSall2). Right: Quantification of P21 protein bands normalized to ACTIN (N = 3, *** P < 0.001 versus shCtrl; Student’s T-test). d) Same analysis as in (b) for Cdkn1a mRNA levels in scramble (shCtrl) versus Sall2-silenced iMEFs (shSall2). We expressed the values as fold change from shCtrl iMEFs (N = 3, *** P < 0.001 versus shCtrl; Student’s T-test). e) Left: Representative Western blot for P21, ANG-mCherry, P53, and ACTIN in Sall2 WT and KO iMEFs. Where indicated, we electroporated plasmids encoding mCherry or ANG-mCherry. After 16 h, the lysates were analyzed. Right: Quantification of P21 protein bands normalized to ACTIN (N = 4 for WT, N = 3 for KO, *** P < 0.001, * P < 0.05, ns = non-significant versus WT or KO; Student’s T-test.). f RNA from Sall2 WT iMEFs electroporated with mCherry or ANG-mCherry for 16 h were isolated, reverse transcribed and analyzed by quantitative real-time PCR. Shown are the expression levels of Cdkn1a normalized to Polr2A (N = 3; data is represented as mean ± s.e.m.). *** P < 0.001 versus mCherry; Student’s T-test. g Model of Cdkn1a regulation based on SALL2 and ANG as opposite regulators. Upper: In the Sall2-silencing model, minor downregulation of the repressor of Cdkn1a (ANG, small blue arrow) and strong downregulation of the activator (SALL2, enlarged blue arrow) downregulates Cdkn1a mRNA. Lower: In the Sall2 KO model, the activator of Cdkn1a (SALL2, red cross) is absent, consequently, the repressor (ANG, enlarged blue arrow) expresses at very low levels, relieving the repression of Cdkn1a mRNA