FIGURE 4.

SALL1 influences the levels of CBX4. (A) Western blot showing protein levels of CBX4-HA when co-expressed with SALL1-YFP or YFP alone in HEK 293FT cells. Actin expression was used as loading control. (B) Western blot showing expression levels of endogenous CBX4 protein in parental HEK 293FT cells (lane 1) or in HEK 293FT cells stably expressing GFS-SALL1 (lane 2). (C) Confocal microscopy images showing inducible expression SALL1-2xHA in HEK 293FT_TripZ-SALL1-2xHA cells. Cells were treated with different concentrations of doxycycline (Dox) to induce SALL1 expression as indicated. SALL1-2xHA was detected using anti-SALL1 primary antibody (green). Cell nuclei were stained with DAPI (blue). (D) Western blot analysis showing expression levels of endogenous CBX4 in HEK 293FT_TripZ-SALL1-2xHA cells treated with increasing concentrations of Dox. (E) Quantification of the expression levels of endogenous CBX4 in HEK 293FT_TripZ-SALL1-2xHA cells treated with increasing concentrations of Dox. Three independent experiments as the one shown in D were performed. The intensity of CBX4 bands was quantified using ImageJ, and the values were normalized to the levels of Actin. P-value was calculated using one-way ANOVA test. *P-value < 0.05. (F) RT-qPCR analysis of SALL1 and CBX4 mRNA expression in HEK 293FT_TripZ-SALL1-2xHA cells treated with increasing concentrations of Dox. SALL1 and CBX4 expression were normalized using GAPDH expression and shown as fold change relative to untreated control. (A,B,D) Molecular weight markers are shown to the right in KDa. Antibodies were used as indicated to the left. (E,F) The mean plus SEM of at least three independent experiments is shown.