Fig. 4: SALL4 cooperates with HDAC1 in reducing GLI1 acetylation levels.

A GLI1-induced transcription has been assessed in HEK293Ts transiently transfected with 12 × GLI1-BS Firefly luciferase, pRL-TK Renilla as normalization control, and the indicated plasmids; data are expressed as FC versus empty vector. Co-IPs of SALL4 and ectopic or endogenous HDAC1 (B, C, respectively), or ectopic or endogenous GLI1 (D, E, respectively) in HEK293Ts transiently transfected as indicated. F Co-IPs of GLI1, SALL4, and HDAC1 in HEK293Ts transiently transfected with vectors expressing the indicated plasmids. G Densitometric analysis of relative GLI1/SALL4 and GLI1/HDAC1 binding affinities, expressed as FC versus relative controls, are normalized to immunoprecipitated Flag-GLI1. H HEK293Ts were transfected with different combinations of indicated plasmids. Protein lysates were immunoprecipitated with anti-Flag agarose beads. One-third of immunocomplexes was probed with antibodies to the indicated proteins (1^st IP), whereas two-thirds were subjected to two elutions with Flag-peptide and re-immunoprecipitated with HA-agarose beads followed by immunoblotting as indicated (2^nd IP). I Co-IP of Flag-GLI1 in HEK293Ts transiently transfected with indicated plasmids. The acetylation levels of GLI1 protein at K518 residue have been assessed by using an in-house generated antibody [39]. Total protein lysates are shown in the Input. J Densitometric analysis of relative AcGLI1 levels, normalized to immunoprecipitated Flag-GLI1. K GLI1-dependent transcription has been assessed in HEK293Ts transiently transfected with 12 × GLI1-BS Firefly luciferase, pRL-TK Renilla as normalization control, and indicated plasmids. Data are expressed as FC versus empty vector. Representative immunoblotting of n = 3 biological replicas with similar results are shown in B–F, H, and I. Densitometric analysis in G and J represent the mean of n = 3 independent experiments ± SD; *p < 0.05 *p < 0.05; **p < 0.01; ***p < 0.001 calculated with two-sided Student’s t-test.